Biodegradable poly(beta-amino esters) and uses thereof

ABSTRACT

Poly(β-amino esters) prepared from the conjugate addition of bis(secondary amines) or primary amines to a bis(acrylate ester) are described. Methods of preparing these polymers from commercially available starting materials are also provided. These tertiary amine-containing polymers are preferably biodegradable and biocompatible and may be used in a variety of drug delivery systems. Given the poly(amine) nature of these polymers, they are particularly suited for the delivery of polynucleotides. Nanoparticles containing polymer/polynucleotide complexes have been prepared. The inventive polymers may also be used to encapsulate other agents to be delivered. They are particularly useful in delivering labile agents given their ability to buffer the pH of their surroundings. A system for preparing and screening polymers in parallel using semi-automated robotic fluid delivery systems is also provided.

RELATED APPLICATIONS

[0001] The present application claims priority under 35 U.S.C. § 120 toand is a continuation-in-part of co-pending application U.S. Ser. No.09/969,431, filed Oct. 2, 2001, entitled “Biodegradable Poly(beta-aminoesters) and Uses Thereof,” which claims priority to provisionalapplications, U.S. S No. 60/305,337, filed Jul. 13, 2001, and U.S. S No.60/239,330, filed Oct. 10, 2000, each of which is incorporated herein byreference.

GOVERNMENT SUPPORT

[0002] The work described herein was supported, in part, by grants fromthe National Science Foundation (Cooperative Agreement #ECC9843342 tothe MIT Biotechnology Process Engineering Center), the NationalInstitutes of Health (GM26698; NRSA Fellowship # 1 F32 GM20227-01), andthe Department of the Army (Cooperative Agreement # DAMD 17-99-2-9-001to the Center for Innovative Minimally Invasive Therapy). The UnitedStates government may have certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] The treatment of human diseases through the application ofnucleotide-based drugs such as DNA and RNA has the potential torevolutionize the medical field (Anderson Nature 392(Suppl.):25-30,1996; Friedman Nature Med. 2:144-147, 1996; Crystal Science 270:404-410,1995; Mulligan Science 260:926-932, 1993; each of which is incorporatedherein by reference). Thus far, the use of modified viruses as genetransfer vectors has generally represented the most clinicallysuccessful approach to gene therapy. While viral vectors are currentlythe most efficient gene transfer agents, concerns surrounding theoverall safety of viral vectors, which include the potential forunsolicited immune responses, have resulted in parallel efforts todevelop non-viral alternatives (for leading references, see: Luo et al.Nat. Biotechnol. 18:33-37,2000; Behr Acc. Chem. Res. 26:274-278, 1993;each of which is incorporated herein by reference). Current alternativesto viral vectors include polymeric delivery systems (Zauner et al. Adv.Drug Del. Rev. 30:97-113, 1998; Kabanov et al. Bioconjugate Chem.6:7-20, 1995; each of which is incorporated herein by reference),liposomal formulations (Miller Angew. Chem. Int. Ed. 37:1768-1785, 1998;Hope et al. Molecular Membrane Technology 15:1-14, 1998; Deshrnukh etal. New J. Chem. 21:113-124, 1997; each of which is incorporated hereinby reference), and “naked” DNA injection protocols (Sanford TrendsBiotechnol. 6:288-302, 1988; incorporated herein by reference). Whilethese strategies have yet to achieve the clinical effectiveness of viralvectors, the potential safety, processing, and economic benefits offeredby these methods (Anderson Nature 392(Suppl.):25-30, 1996; incorporatedherein by reference) have ignited interest in the continued developmentof non-viral approaches to gene therapy (Boussif et al. Proc. Natl.Acad. Sci. USA 92:7297-7301, 1995; Putnam et al. Macromolecules32:3658-3662, 1999; Lim et al. J. Am. Chem. Soc. 121:5633-5639, 1999;Gonzalez et al. Bioconjugate Chem. 10:1068-1074, 1999; Kukowska-Latalloet al. Proc. Natl. Acad. Sci. USA 93:4897-4902, 1996; Tang et al.Bioconjugate Chem. 7:703-714, 1996; Haensler et al. Bioconjugate Chem.4:372-379, 1993; each of which is incorporated herein by reference).

[0004] Cationic polymers have been widely used as transfection vectorsdue to the facility with which they condense and protect negativelycharged strands of DNA. Amine-containing polymers such as poly(lysine)(Zauner et al. Adv. Drug Del. Rev. 30:97-113, 1998; Kabanov et al.Bioconjugate Chem. 6:7-20, 1995; each of which is incorporated herein byreference), poly(ethylene imine) (PEI) (Boussif et al. Proc. Natl. Acad.Sci. USA 92:7297-7301, 1995; incorporated herein by reference), andpoly(amidoamine) dendrimers (Kukowska-Latallo et al. Proc. Natl. Acad.Sci. USA 93:4897-4902, 1996; Tang et al. Bioconjugate Chem. 7:703-714,1996; Haensler et al. Bioconjugate Chem. 4:372-379, 1993; each of whichis incorporated herein by reference) are positively-charged atphysiological pH, form ion pairs with nucleic acids, and mediatetransfection in a variety of cell lines. Despite their common use,however, cationic polymers such as poly(lysine) and PEI can besignificantly cytotoxic (Zauner et al. Adv. Drug Del. Rev. 30:97-113,1998; Deshmukh et al. New J. Chem. 21:113-124, 1997; Choksakulnimitr etal. Controlled Release 34:233-241, 1995; Brazeau et al. Pharm. Res.15:680-684, 1998; each of which is incorporated herein by reference). Asa result, the choice of cationic polymer for a gene transfer applicationgenerally requires a trade-off between transfection efficiency andshort- and long-term cytotoxicity. Additionally, the long-termbiocompatibility of these polymers remains an important issue for use intherapeutic applications in vivo, since several of these polymers arenot readily biodegradable (Uhrich Trends Polym. Sci. 5:388-393, 1997;Roberts et al. J. Biomed. Mater. Res. 30:53-65, 1996; each of which isincorporated herein by reference).

[0005] In order to develop safe alternatives to existing polymericvectors and other functionalized biomaterials, degradable polyestersbearing cationic side chains have been developed (Putnam et al.Macromolecules 32:3658-3662, 1999; Barrera et al. J. Am. Chem. Soc.115:11010-11011, 1993; Kwon et al. Macromolecules 22:3250-3255, 1989;Lim et al. J. Am. Chem. Soc. 121:5633-5639, 1999; Zhou et al.Macromolecules 23:3399-3406, 1990; each of which is incorporated hereinby reference). Examples of these polyesters includepoly(L-lactideco-L-lysine) (Barrera et al. J. Am. Chem. Soc.115:11010-11011, 1993; incorporated herein by reference), poly(serineester) (Zhou et al. Macromolecules 23:3399-3406, 1990; each of which isincorporated herein by reference), poly(4-hydroxy-L-proline ester)(Putnam et al. Macromolecules 32:3658-3662, 1999; Lim et al. J. Am.Chem. Soc. 121:5633-5639, 1999; each of which is incorporated herein byreference), and more recently, poly[α-(4-aminobutyl)-L-glycolic acid].Poly(4-hydroxy-L-proline ester) and poly[α-(4-aminobutyl)-L-glycolicacid] were recently demonstrated to condense plasmid DNA throughelectrostatic interactions, and to mediate gene transfer (Putnam et al.Macromolecules 32:3658-3662, 1999; Lim et al. J. Am. Chem. Soc.121:5633-5639, 1999; each of which is incorporated herein by reference).Importantly, these new polymers are significantly less toxic thanpoly(lysine) and PEI, and they degrade into non-toxic metabolites. It isclear from these investigations that the rational design ofamine-containing polyesters can be a productive route to the developmentof safe, effective transfection vectors. Unfortunately, however, presentsyntheses of these polymers require either the independent preparationof specialized monomers (Barrera et al. J. Am. Chem. Soc.115:11010-11011, 1993; incorporated herein by reference), or the use ofstoichiometric amounts of expensive coupling reagents (Putnam et al.Macromolecules 32:3658-3662, 1999; incorporated herein by reference).Additionally, the amine functionalities in the monomers must beprotected prior to polymerization (Putnam et al. Macromolecules32:3658-3662, 1999; Lim et al. J. Am. Chem. Soc. 121:5633-5639, 1999;Gonzalez et al. Bioconjugate Chem. 10: 1068-1074, 1999; Barrera et al.J. Am. Chem. Soc. 115:11010-11011, 1993; Kwon et al. Macromolecules22:3250-3255, 1989; each of which is incorporated herein by reference),necessitating additional post-polymerization deprotection steps beforethe polymers can be used as transfection agents.

[0006] There exists a continuing need for non-toxic, biodegradable,biocompatible polymers that can be used to transfect nucleic acids andthat are easily prepared efficiently and economically. Such polymerswould have several uses, including the delivery of nucleic acids in genetherapy as well as in the packaging and/or delivery of diagnostic,therapeutic, and prophylactic agents.

SUMMARY OF THE INVENTION

[0007] The present invention provides polymers useful in preparing drugdelivery devices and pharmaceutical compositions thereof. The inventionalso provides methods of preparing the polymers and methods of preparingmicrospheres and other pharmaceutical compositions containing theinventive polymers.

[0008] The polymers of the present invention are poly(βamino esters) andsalts and derivatives thereof. Preferred polymers are biodegradable andbiocompatible. Typically, the polymers have one or more tertiary aminesin the backbone of the polymer. Preferred polymers have one or twotertiary amines per repeating backbone unit. The polymers may also beco-polymers in which one of the components is a poly(β-amino ester). Thepolymers of the invention may readily be prepared by condensingbis(secondary amines) or primary amines with bis(acrylate esters). Apolymer of the invention is represented by either of the formulae below:

[0009] where A and B are linkers which may be any substituted orunsubstituted, branched or unbranched chain of carbon atoms orheteroatoms. The molecular weights of the polymers may range from 1000g/mol to 20,000 g/mol, preferably from 5000 g/mol to 15,000 g/mol. Incertain embodiments, B is an alkyl chain of one to twelve carbons atoms.In other embodiments, B is a heteroaliphatic chain containing a total ofone to twelve carbon atoms and heteroatoms. The groups R₁ and R₂ may beany of a wide variety of substituents. In certain embodiments, R₁ and R₂may contain primary amines, secondary amines, tertiary amines, hydroxylgroups, and alkoxy groups. In certain embodiments, the polymers areamine-terminated; and in other embodiments, the polymers are acrylatedterminated. In a particularly preferred embodiment, the groups R₁ and/orR₂ form cyclic structures with the linker A (see the DetailedDescription section below). Polymers containing such cyclic moietieshave the characteristic of being more soluble at lower pH. Specificallypreferred polymers are those that are insoluble in aqueous solutions atphysiologic pH (pH 7.2-7.4) and are soluble in aqueous solutions belowphysiologic pH (pH<7.2). Other preferred polymers are those that aresoluble in aqueous solution at physiologic pH (pH 7.2-7.4) and below.Preferred polymers are useful in the transfection of polynucleotidesinto cells.

[0010] In another aspect, the present invention provides a method ofpreparing the inventive polymers. In a preferred embodiment,commercially available or readily available monomers, bis(secondaryamines), primary amines, and bis(acrylate esters), are subjected toconditions which lead to the conjugate addition of the amine to thebis(acrylate ester). In a particularly preferred embodiment, each of themonomers is dissolved in an organic solvent (e.g., DMSO, DMF, THF,diethyl ether, methylene chloride, hexanes, etc.), and the resultingsolutions are combined and heated for a time sufficient to lead topolymerization of the monomers. In other embodiments, the polymerizationis carried out in the absence of solvent. The resulting polymer may thenbe purified and optionally characterized using techniques known in theart.

[0011] In yet another aspect of the invention, the polymers are used toform nanometer-scale complexes with nucleic acids. Thepolynucleotide/polymer complexes may be formed by adding a solution ofpolynucleotide to a vortexing solution of the polymer at a desiredDNA/polymer concentration. The weight to weight ratio of polynucleotideto polymer may range from 1:0.1 to 1:200, preferably from 1:10 to 1:150,more preferably from 1:50 to 1:150. The amine monomer to polynucleotidephosphate ratio may be approximately 10: 1, 15:1, 20:1, 25:1, 30:1,35:1, 40: 1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1,95:1, 100:1, 110:1, 120:1, 130:1, 140:1, 150:1, 160:1, 170:1, 180:1,190:1, and 200:1. The cationic polymers condense the polynucleotide intosoluble particles typically 50-500 nm in size. Thesepolynucleotide/polymer complexes may be used in the delivery ofpolynucleotides to cells. In a particularly preferred embodiment, thesecomplexes are combined with pharmaceutical excipients to formpharmaceutical compositions suitable for delivery to animals includinghumans. In certain embodiments, a polymer with a high molecular weightto nitrogen atom ratio (e.g., polylysine, polyethyleneimine) is used toincrease transfection efficiency.

[0012] In another aspect of the invention, the polymers are used toencapsulate therapeutic, diagnostic, and/or prophylactic agentsincluding polynucleotides to form microparticles. Typically thesemicroparticles are an order of magnitude larger than thepolynucleotide/polymer complexes. The microparticles range from 1micrometer to 500 micrometers. In a particularly preferred embodiment,these microparticles allow for the delivery of labile small molecules,proteins, peptides, and/or polynucleotides to an individual. Themicroparticles may be prepared using any of the techniques known in theart to make microparticles, such as, for example, double emulsion, phaseinversion, and spray drying. In a particularly preferred embodiment, themicroparticles can be used for pH-triggered delivery of the encapsulatedcontents due to the pH-responsive nature of the polymers (i.e., beingmore soluble at lower pH).

[0013] In yet another aspect, the invention provides a system forsynthesizing and screening a collection of polymers. In certainembodiments, the system takes advantage of techniques known in the artof automated liquid handling and robotics. The system of synthesizingand screening may be used with poly(beta-amino ester)s as well as othertypes of polymers including polyamides, polyesters, polyethers,polycarbamates, polycarbonates, polyureas, polyamines, etc. Thecollection of polymers may be a collection of all one type of polymer(e.g., all poly(betamino esters) or may be a diverse collection ofpolymers. Thousands of polymers may be synthesized and screened inparallel using the inventive system. In certain embodiments, thepolymers are screened for properties useful in the field of genedelivery, transfection, or gene therapy. Some of these propertiesinclude solubility at various pHs, ability to complex polynucleotides,ability to transfect a polynucleotide into a cell, etc. Certainpoly(beta-amino ester)s found to be useful in transfecting cells includeM17, KK89, C32, C36, JJ28, JJ32, JJ36, LL6, and D94 as described inExamples 4 and 5.

Definitions

[0014] The following are chemical terms used in the specification andclaims:

[0015] The term acyl as used herein refers to a group having the generalformula —C(═O)R, where R is alkyl, alkenyl, alkynyl, aryl, carbocylic,heterocyclic, or aromatic heterocyclic. An example of an acyl group isacetyl.

[0016] The term alkyl as used herein refers to saturated, straight- orbranched-chain hydrocarbon radicals derived from a hydrocarbon moietycontaining between one and twenty carbon atoms by removal of a singlehydrogen atom. In some embodiments, the alkyl group employed in theinvention contains 1-10 carbon atoms. In another embodiment, the alkylgroup employed contains 1-8 carbon atoms. In still other embodiments,the alkyl group contains 1-6 carbon atoms. In yet another embodiments,the alkyl group contains 1-4 carbons. Examples of alkyl radicalsinclude, but are not limited to, methyl, ethyl, n-propyl, isopropyl,n-butyl, iso-butyl, sec-butyl, sec-pentyl, iso-pentyl, tert-butyl,n-pentyl, neopentyl, n-hexyl, sec-hexyl, n-heptyl, n-octyl, n-decyl,n-undecyl, dodecyl, and the like, which may bear one or moresustitutents.

[0017] The term alkoxy as used herein refers to a saturated (i.e.,alkyl-O—) or unsaturated (i.e., alkenyl-O— and alkynyl-O—) groupattached to the parent molecular moiety through an oxygen atom. Incertain embodiments, the alkyl group contains 1-20 aliphatic carbonatoms. In certain other embodiments, the akyl, akenyl, and alkynylgroups employed in the invention contain 1-8 aliphatic carbon atoms. Instill other embodiments, the alkyl group contains 1-6 aliphatic carbonatoms. In yet other embodiments, the alkyl group contains 1-4 aliphaticcarbon atoms. Examples include, but are not limited to, methoxy, ethoxy,propoxy, isopropoxy, n-butoxy, tert-butoxy, i-butoxy, sec-butoxy,neopentoxy, n-hexoxy, and the like.

[0018] The term alkenyl denotes a monovalent group derived from ahydrocarbon moiety having at least one carbon-carbon double bond by theremoval of a single hydrogen atom. In certain embodiments, the alkenylgroup employed in the invention contains 1-20 carbon atoms. In someembodiments, the alkenyl group employed in the invention contains 1-10carbon atoms. In another embodiment, the alkenyl group employed contains1-8 carbon atoms. In still other embodiments, the alkenyl group contains1-6 carbon atoms. In yet another embodiments, the alkenyl group contains1-4 carbons. Alkenyl groups include, for example, ethenyl, propenyl,butenyl, 1-methyl-2-buten-1-yl, and the like.

[0019] The term alkynyl as used herein refers to a monovalent groupderived form a hydrocarbon having at least one carbon-carbon triple bondby the removal of a single hydrogen atom. In certain embodiments, thealkynyl group employed in the invention contains 1-20 carbon atoms. Insome embodiments, the alkynyl group employed in the invention contains1-10 carbon atoms. In another embodiment, the alkynyl group employedcontains 1-8 carbon atoms. In still other embodiments, the alkynyl groupcontains 1-6 carbon atoms. Representative alkynyl groups include, butare not limited to, ethynyl, 2-propynyl (propargyl), 1-propynyl, and thelike.

[0020] The term alkylamino, dialkylamino, and trialkylamino as usedherein refers to one, two, or three, respectively, alkyl groups, aspreviously defined, attached to the parent molecular moiety through anitrogen atom. The term alkylamino refers to a group having thestructure—NHR′ wherein R′ is an alkyl group, as previously defined; andthe term dialkylamino refers to a group having the structure —NR′R″,wherein R′ and R′ are each independently selected from the groupconsisting of alkyl groups. The term trialkylamino refers to a grouphaving the structure —NR′R″R′″, wherein R′, R″, and R′″ are eachindependently selected from the group consisting of alkyl groups. Incertain embodiments, the alkyl group contain 1-20 aliphatic carbonatoms. In certain other embodiments, the alkyl group contains 1-10aliphatic carbon atoms. In yet other embodiments, the alkyl groupcontains 1-8 aliphatic carbon atoms. In still other embodiments, thealkyl group contain 1-6 aliphatic carbon atoms. In yet otherembodiments, the alkyl group contain 1-4 aliphatic carbon atoms.Additionally, R′, R″, and/or R′″ taken together may optionally be—(CH₂)_(k)— where k is an integer from 2 to 6. Examples include, but arenot limited to, methylamino, dimethylamino, ethylamino, diethylamino,diethylaminocarbonyl, methylethylamino, iso-propylamino, piperidino,trimethylamino, and propylamino.

[0021] The terms alkylthioether and thioalkoxyl refer to a saturated(i.e., alkyl-S—) or unsaturated (i.e., alkenyl-S— and alkynyl-S—) groupattached to the parent molecular moiety through a sulfur atom. Incertain embodiments, the alkyl group contains 1-20 aliphatic carbonatoms. In certain other embodiments, the alkyl group contains 1-10aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl,and alkynyl groups contain 1-8 aliphatic carbon atoms. In still otherembodiments, the alkyl, alkenyl, and alkynyl groups contain 1-6aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl,and alkynyl groups contain 1-4 aliphatic carbon atoms. Examples ofthioalkoxyl moieties include, but are not limited to, methylthio,ethylthio, propylthio, isopropylthio, n-butylthio, and the like.

[0022] The term aryl as used herein refers to an unsaturated cyclicmoiety comprising at least one aromatic ring. In certain embodiments,aryl group refers to a mono- or bicyclic carbocyclic ring system havingone or two aromatic rings including, but not limited to, phenyl,naphthyl, tetrahydronaphthyl, indanyl, indenyl, and the like. Arylgroups can be unsubstituted or substituted with substituents selectedfrom the group consisting of branched and unbranched alkyl, alkenyl,alkynyl, haloalkyl, alkoxy, thioalkoxy, amino, alkylamino, dialkylamino,trialkylamino, acylamino, cyano, hydroxy, halo, mercapto, nitro,carboxyaldehyde, carboxy, alkoxycarbonyl, and carboxamide. In addition,substituted aryl groups include tetrafluorophenyl and pentafluorophenyl.

[0023] The term carboxylic acid as used herein refers to a group offormula —CO₂H.

[0024] The terms halo and halogen as used herein refer to an atomselected from fluorine, chlorine, bromine, and iodine.

[0025] The term heterocyclic, as used herein, refers to an aromatic ornon-aromatic, partially unsaturated or fully saturated, 3- to10-membered ring system, which includes single rings of 3 to 8 atoms insize and bi- and tri-cyclic ring systems which may include aromaticfive- or six-membered aryl or aromatic heterocyclic groups fused to anon-aromatic ring. These heterocyclic rings include those having fromone to three heteroatoms independently selected from oxygen, sulfur, andnitrogen, in which the nitrogen and sulfur heteroatoms may optionally beoxidized and the nitrogen heteroatom may optionally be quaternized. Incertain embodiments, the term heterocylic refers to a non-aromatic 5-,6-, or 7-membered ring or a polycyclic group wherein at least one ringatom is a heteroatom selected from O, S, and N (wherein the nitrogen andsulfur heteroatoms may be optionally oxidized), including, but notlimited to, a bi- or tri-cyclic group, comprising fused six-memberedrings having between one and three heteroatoms independently selectedfrom the oxygen, sulfur, and nitrogen, wherein (i) each 5-membered ringhas 0 to 2 double bonds, each 6-membered ring has 0 to 2 double bonds,and each 7-membered ring has 0 to 3 double bonds, (ii) the nitrogen andsulfur heteroatoms may be optionally oxidized, (iii) the nitrogenheteroatom may optionally be quaternized, and (iv) any of the aboveheterocyclic rings may be fused to an aryl or heteroaryl ring.

[0026] The term aromatic heterocyclic, as used herein, refers to acyclic aromatic radical having from five to ten ring atoms of which onering atom is selected from sulfur, oxygen, and nitrogen; zero, one, ortwo ring atoms are additional heteroatoms independently selected fromsulfur, oxygen, and nitrogen; and the remaining ring atoms are carbon,the radical being joined to the rest of the molecule via any of the ringatoms, such as, for example, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl,pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl,oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, and thelike. Aromatic heterocyclic groups can be unsubstituted or substitutedwith substituents selected from the group consisting of branched andunbranched alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, thioalkoxy,amino, alkylamino, dialkylamino, trialkylamino, acylamino, cyano,hydroxy, halo, mercapto, nitro, carboxyaldehyde, carboxy,alkoxycarbonyl, and carboxamide.

[0027] Specific heterocyclic and aromatic heterocyclic groups that maybe included in the compounds of the invention include:3-methyl-4-(3-methylphenyl)piperazine, 3 methylpiperidine,4-(bis-(4-fluorophenyl)methyl)piperazine, 4-(diphenylmethyl)piperazine,4(ethoxycarbonyl)piperazine, 4-(ethoxycarbonylmethyl)piperazine,4-(phenylmethyl)piperazine, 4-(1-phenylethyl)piperazine,4-(1,1-dimethylethoxycarbonyl)piperazine, 4-(2-(bis-(2-propenyl)amino)ethyl)piperazine, 4-(2-(diethylamino)ethyl)piperazine,4-(2-chlorophenyl)piperazine, 4(2-cyanophenyl)piperazine,4-(2-ethoxyphenyl)piperazine, 4-(2-ethylphenyl)piperazine,4-(2-fluorophenyl)piperazine, 4-(2-hydroxyethyl)piperazine,4-(2-methoxyethyl)piperazine, 4-(2-methoxyphenyl)piperazine,4-(2-methylphenyl)piperazine, 4-(2-methylthiophenyl) piperazine,4(2-nitrophenyl)piperazine, 4-(2-nitrophenyl)piperazine,4-(2-phenylethyl)piperazine, 4-(2-pyridyl)piperazine,4-(2-pyrimidinyl)piperazine, 4-(2,3-dimethylphenyl)piperazine,4-(2,4-difluorophenyl) piperazine, 4-(2,4-dimethoxyphenyl)piperazine,4-(2,4-dimethylphenyl)piperazine, 4-(2,5-dimethylphenyl)piperazine,4-(2,6-dimethylphenyl)piperazine, 4-(3-chlorophenyl)piperazine,4-(3-methylphenyl)piperazine, 4-(3-trifluoromethylphenyl)piperazine,4-(3,4-dichlorophenyl)piperazine, 4-3,4-dimethoxyphenyl)piperazine,4-(3,4-dimethylphenyl)piperazine,4-(3,4-methylenedioxyphenyl)piperazine,4-(3,4,5-trimethoxyphenyl)piperazine, 4-(3,5-dichlorophenyl)piperazine,4-(3,5-dimethoxyphenyl)piperazine,4-(4-(phenylmethoxy)phenyl)piperazine,4-(4-(3,1-dimethylethyl)phenylmethyl)piperazine,4-(4-chloro-3-trifluoromethylphenyl)piperazine,4-(4-chlorophenyl)-3-methylpiperazine, 4-(4-chlorophenyl)piperazine,4-(4-chlorophenyl)piperazine, 4-(4-chlorophenylmethyl)piperazine,4-(4-fluorophenyl)piperazine, 4-(4-methoxyphenyl)piperazine,4-(4-methylphenyl)piperazine, 4-(4-nitrophenyl)piperazine,4-(4-trifluoromethylphenyl)piperazine, 4-cyclohexylpiperazine,4-ethylpiperazine, 4-hydroxy-4-(4-chlorophenyl)methylpiperidine,4-hydroxy-4-phenylpiperidine, 4-hydroxypyrrolidine, 4-methylpiperazine,4-phenylpiperazine, 4-piperidinylpiperazine,4-(2-furanyl)carbonyl)piperazine,4-((1,3-dioxolan-5-yl)methyl)piperazine,6-fluoro-1,2,3,4-tetrahydro-2-methylquinoline, 1,4-diazacylcloheptane,2,3-dihydroindolyl, 3,3-dimethylpiperidine, 4,4-ethylenedioxypiperidine,1,2,3,4-tetrahydroisoquinoline, 1,2,3,4-tetrahydroquinoline,azacyclooctane, decahydroquinoline, piperazine, piperidine, pyrrolidine,thiomorpholine, and triazole.

[0028] The term carbamoyl, as used herein, refers to an amide group ofthe formula —CONH₂.

[0029] The term carbonyldioxyl, as used herein, refers to a carbonategroup of the formula —O—CO—OR.

[0030] The term hydrocarbon, as used herein, refers to any chemicalgroup comprising hydrogen and carbon. The hydrocarbon may be substitutedor unsubstitued. The hydrocarbon may be unsaturated, saturated,branched, unbranched, cyclic, polycyclic, or heterocyclic. Illustrativehydrocarbons include, for example, methyl, ethyl, n-propyl, iso-propyl,cyclopropyl, allyl, vinyl, n-butyl, tert-butyl, ethynyl, cyclohexyl,methoxy, diethylamino, and the like. As would be known to one skilled inthis art, all valencies must be satisfied in making any substitutions.

[0031] The terms substituted, whether preceded by the term “optionally”or not, and substituent, as used herein, refer to the ability, asappreciated by one skilled in this art, to change one functional groupfor another functional group provided that the valency of all atoms ismaintained. When more than one position in any given structure may besubstituted with more than one substituent selected from a specifiedgroup, the substituent may be either the same or different at everyposition. The substituents may also be further substituted (e.g., anaryl group substituent may have another substituent off it, such asanother aryl group, which is further substituted with fluorine at one ormore positions).

[0032] The term thiohydroxyl or thiol, as used herein, refers to a groupof the formula —SH.

[0033] The term ureido, as used herein, refers to a urea group of theformula —NH—CO—NH₂.

[0034] The following are more general terms used throughout the presentapplication:

[0035] “Animal”: The term animal, as used herein, refers to humans aswell as non-human animals, including, for example, mammals, birds,reptiles, amphibians, and fish. Preferably, the non-human animal is amammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, acat, a primate, or a pig). An animal may be a domesticated animal. Ananimal may be a transgenic animal.

[0036] “Associated with”: When two entities are “associated with” oneanother as described herein, they are linked by a direct or indirectcovalent or non-covalent interaction. Preferably, the association iscovalent. Desirable non-covalent interactions include hydrogen bonding,van der Waals interactions, hydrophobic interactions, magneticinteractions, electrostatic interactions, etc.

[0037] “Biocompatible”: The term “biocompatible”, as used herein isintended to describe compounds that are not toxic to cells. Compoundsare “biocompatible” if their addition to cells in vitro results in lessthan or equal to 20% cell death, and their administration in vivo doesnot induce inflammation or other such adverse effects.

[0038] “Biodegradable”: As used herein, “biodegradable” compounds arethose that, when introduced into cells, are broken down by the cellularmachinery or by hydrolysis into components that the cells can eitherreuse or dispose of without significant toxic effect on the cells (i.e.,fewer than about 20% of the cells are killed when the components areadded to cells in vitro). The components preferably do not induceinflammation or other adverse effects in vivo. In certain preferredembodiments, the chemical reactions relied upon to break down thebiodegradable compounds are uncatalyzed.

[0039] “Effective amount”: In general, the “effective amount” of anactive agent or drug delivery device refers to the amount necessary toelicit the desired biological response. As will be appreciated by thoseof ordinary skill in this art, the effective amount of an agent ordevice may vary depending on such factors as the desired biologicalendpoint, the agent to be delivered, the composition of theencapsulating matrix, the target tissue, etc. For example, the effectiveamount of microparticles containing an antigen to be delivered toimmunize an individual is the amount that results in an immune responsesufficient to prevent infection with an organism having the administeredantigen.

[0040] “Peptide” or “protein”: According to the present invention, a“peptide” or “protein” comprises a string of at least three amino acidslinked together by peptide bonds. The terms “protein” and “peptide” maybe used interchangeably. Peptide may refer to an individual peptide or acollection of peptides. Inventive peptides preferably contain onlynatural amino acids, although non-natural amino acids (i.e., compoundsthat do not occur in nature but that can be incorporated into apolypeptide chain) and/or amino acid analogs as are known in the art mayalternatively be employed. Also, one or more of the amino acids in aninventive peptide may be modified, for example, by the addition of achemical entity such as a carbohydrate group, a phosphate group, afarnesyl group, an isofarnesyl group, a fatty acid group, a linker forconjugation, functionalization, or other modification, etc. In apreferred embodiment, the modifications of the peptide lead to a morestable peptide (e.g., greater half-life in vivo). These modificationsmay include cyclization of the peptide, the incorporation of D-aminoacids, etc. None of the modifications should substantially interferewith the desired biological activity of the peptide.

[0041] “Polynucleotide” or “oligonucleotide”: Polynucleotide oroligonucleotide refers to a polymer of nucleotides. Typically, apolynucleotide comprises at least three nucleotides. The polymer mayinclude natural nucleosides (i.e., adenosine, thymidine, guanosine,cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, anddeoxycytidine), nucleoside analogs (e.g., 2-aminoadenosine,2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine,C5-propynylcytidine, C5-propynyluridine, C5-bromouridine,C5-fluorouridine, C5-iodouridine, C5-methylcytidine, 7-deazaadenosine,7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine,and 2-thiocytidine), chemically modified bases, biologically modifiedbases (e.g., methylated bases), intercalated bases, modified sugars(e.g., 2′-fluororibose, ribose, 2′-deoxyribose, arabinose, and hexose),or modified phosphate groups (e.g., phosphorothioates and5′-N-phosphoramidite linkages).

[0042] “Small molecule”: As used herein, the term “small molecule”refers to organic compounds, whether naturally-occurring or artificiallycreated (e.g., via chemical synthesis) that have relatively lowmolecular weight and that are not proteins, polypeptides, or nucleicacids. Typically, small molecules have a molecular weight of less thanabout 1500 g/mol. Also, small molecules typically have multiplecarbon-carbon bonds. Known naturally-occurring small molecules include,but are not limited to, penicillin, erythromycin, taxol, cyclosporin,and rapamycin. Known synthetic small molecules include, but are notlimited to, ampicillin, methicillin, sulfamethoxazole, and sulfonamides.

BRIEF DESCRIPTION OF THE DRAWING

[0043]FIG. 1 shows the time profile for the degradation of polymers 1-3at 37° C. at pH 5.1 and pH 7.4. Degradation is expressed as percentdegradation over time based on GPC data.

[0044]FIG. 2 shows cytotoxicity profiles of polymers 1-3 and PEI.Viability of NIH 3T3 cells is expressed as a function of polymerconcentration. The molecular weights of polymers 1, 2, and 3 were 5800,11300, and 22500, respectively. The molecular weight of the PEI employedwas 25000.

[0045]FIG. 3 shows the retardation of pCMV-Luc DNA by polymer 1 inagarose gel electrophoresis. Each lane corresponds to a differentDNA/polymer weight ratio. The ratios are as follows: 1) 1:0 (DNA only);2) 1:0.5; 3) 1:1; 4) 1:2; 5) 1:3; 6) 1:4; 7) 1:5; 8) 1:6; 9) 1:7; and10) 1:8.

[0046]FIG. 4 shows the average effective diameters of DNA/polymercomplexes formed from pCMV-Luc plasmid and polymer 3 (M_(n)=31,000) as afunction of polymer concentration.

[0047]FIG. 5 shows average ζ-potentials of DNA/polymer complexes formedfrom pCMV-Luc plasmid and polymer 3 (M_(n)=31,000) as a function ofpolymer concentration. The numbers for each complex correspond to thecomplex numbers in FIG. 4.

[0048]FIG. 6 is an SEM image of rhodamine/dextran-loaded microspheresfabricated from polymer 1.

[0049]FIG. 7 shows the release profiles of rhodamine/dextran frompolymer 1 and PLGA microspheres at various pH values. The arrowsindicate the points at which HEPES buffer (pH 7.4) was exchanged withacetate buffer (pH 5.1).

[0050]FIG. 8 shows a) a representative fluorescence microscopy image ofrhodamine/dextran-loaded polymer 1 microspheres suspended in HEPESbuffer (pH 7.4). FIG. 8b shows a sample of loaded polymer 1 microspheresat pH 7.4 after addition of acetate buffer (pH 5.1). The direction ofdiffusion of acid is from the top right to the bottom left of the image(elapsed time seconds).

[0051]FIG. 9 demonstrates the gel electrophoresis assay used to identifyDNA-complexing polymers. Lane annotations correspond to the 70water-soluble members of the screening library. For each polymer, assayswere performed at DNA/polymer ratios of 1:5 (left well) and 1:20 (rightwell). Lanes marked C* contain DNA alone (no polymer) and were used as acontrol.

[0052]FIG. 10 shows transfection data as a function of structure for anassay employing pCMV-Luc (600 ng/well, DNA/polymer=1:20). Light unitsare arbitrary and not normalized to total cell protein; experiments wereperformed in triplicate (error bars not shown). Black squares representwater-insoluble polymers, white squares represent water-soluble polymersthat did not complex DNA in FIG. 9. The right column (marked “^(*)”)displays values for the following control experiments: no polymer(green), PEI (red), and Lipofectamine (light blue).

[0053]FIG. 11 shows a synthesis of poly(beta-amino ester)s.Poly(beta-amino ester)s may be synthesized by the conjugate addition ofprimary amines (equation 1) or bis(secondary amines) (equation 2) todiacrylates.

[0054]FIG. 12 shows a variety of amine (A) and diacrylate (B) monomersused in the synthesis of the polymer library.

[0055]FIG. 13 is a histogram of polymer transfection efficiencies. Inthe first screen all 2350 polymers were tested for their ability todeliver pCMV-luc DNA at N:P ratios of 40:1, 20: 1, and 10:1 to COS-7cells. Transfection efficiency is presented in ng Luciferase per well.For reference, PEI transfection efficiency is shown. COS-7 cells readilytake up naked DNA, and in our conditions produce 0.15±0.05 ng per well,and the lipid reagent, Lipofectamine 2000, produces 13.5±1.9 ng perwell.

[0056]FIG. 14. A) Optimized transfection efficiency of the top 50polymers relative to PEI and lipofectamine 2000. Polymers were tested asdescribed in methods. In the first broad screen N:P ratios of 40:1, 20:1, and 10:1 with an n of 1 were tested. The top 93 were rescreened atsix different N:P ratios=(optimal N:P form the first screen)×1.75, 1.5,1.25, 1.0, 0.75, and 0.5, in triplicate. Control reactions are labeledin Red, and polymers that did not bind DNA in a gel electrophoresisassay are shown in black. B) DNA binding polymers as determined byagarose gel electrophoresis. The data was tabulated in the followingmanner: 1) fully shifted DNA is represented by (+), 2) partially shiftedDNA is represented by (+/−), 3) unbound DNA is represented by (−).

[0057]FIG. 15 shows the transfection of COS-7 cells using enhanced GreenFluorescent Protein plasmid. Cells were transfected at an N:P ratio of(optimal N:P from the broad screen)×1.25 with 600 ng of DNA. Regions ofthe well showing high transfection are shown for the following polymers:a) C36, b) D94.

[0058]FIG. 16 shows how the polymer molecular weight and the chainend-group is affected by varying the amine/diacrylate ratio in thereaction mixture. Molecular weights (Mw) (relative to polystyrenestandards) were determined by organic phase GPC. Polymers synthesizedwith amine/diacrylate ratios of >1 have amine end-groups, and polymerssynthesized with amine/diacrylate ratios of <1 have acrylate end-groups.

[0059]FIG. 17 shows luciferase transfection results for Poly-1 as afunction of polymer molecular weight, polymer/DNA ratio (w/w), andpolymer end-group. (A) amine-terminated chains; (B) acrylate-terminatedchains. (n=4, error bars are not shown.)

[0060]FIG. 18 shows luciferase transfection results for Poly-2 as afunction of polymer molecular weight, polymer/DNA ratio (w/w), andpolymer end-group. (A) amine-terminated chains; (B) acrylate-terminatedchains. (n—4, error bars not shown).

[0061]FIG. 19 shows the cytotoxicity of poly-1/DNA complexes as afunction of polymer molecular weight, polymer/DNA ratio (w/w), andpolymer end-group. (A) amine-terminated chains; (B) acrylate-terminatedchains. (n=4, error bars are not shown.)

[0062]FIG. 20 shows the cytotoxicity of poly-2/DNA complexes as afunction of polymer molecular weight, polymer/DNA ratio (w/w), andpolymer end-group. (A) amine-terminated chains; (B) acrylate-terminatedchains. (n=4, error bars are not shown.)

[0063]FIG. 21 shows the relative cellular uptake level of poly-1/DNAcomplexes as a function of polymer molecular weight, polymer/DNA ratio(w/w), and polymer end-group. (A) amine-terminated chains; (B)acrylate-terminated chains. (n=4, error bars are not shown.)

[0064]FIG. 22 shows the relative cellular uptake level of poly-2/DNAcomplexes as a function of polymer molecular weight, polymer/DNA ratio(w/w), and polymer end-group. (A) amine-terminated chains (blank squaresrepresent conditions where cytotoxicity of the complexes prevented areliable measurement of cellular uptake); (B) acrylate-terminatedchains. (n=4, error bars not shown.)

[0065]FIG. 23 shows the enhancement of transfection activity of poly-1(amine-terminated chains, M_(W)=13,100) based delivery vectors throughthe use of co-complexing agents. (A) polylysine (PLL); (B)polyethyleneimine (PEI). (n=4, error bars are not shown).

[0066]FIG. 24 shows the enhancement of transfection activity of poly-2(amine-terminated chains, MW=13,400) based delivery vectors through theuse of co-complexing agents. (A) polylysine (PLL); (B) polyethyleneimine(PEI). (n=4, error bars are not shown.)

[0067]FIG. 25 is a comparison of GFP gene transfer into COS-7 cellsusing Poly-1/PLL (Poly-1:PLL:DNA=60:0.1:1 (w/w/w)), Poly-2/PLL(Poly-2:PLL:DNA=15:0.4:1 (w/w/w)), Lipofectamine 2000 (μL reagent: μgDNA=1:1), PEI (PEI:DNA 1:1 (w/w), N/P 8), and naked DNA. Cells wereseeded on 6-well plates and grown to new confluence. Cells were theincubated with complexes (5 μg DNA/well) for 1 hour, after which timecomplexes were removed and fresh growth media was added. Two days laterGFP expression was assayed by flow cytometry. (n=3, error bars indicateone standard deviation.)

[0068]FIG. 26 shows GFP expression in COS-7 cells transfected usingPoly-1/PLL.

[0069]FIG. 27 shows GFP gene transfer into four different cell linesusing Poly-1/PLL (Poly-1:PLL:DNA=60:0.1:1 (w/wlw). Cells were seeded on6-well plates and grown to near confluence. Cells were then incubatedwith complexes (5 μg DNA/well) for 1 hour, after which time complexeswere removed and fresh growth media was added. Two days later GFPexpression was assayed by flow cytometry. (n=5, error bars indicate onestandard deviation.)

DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS OF THE INVENTION

[0070] The present invention provides improved polymeric encapuslationand delivery systems based on the use of β-amino ester polymers. Thesytems may be used in the pharmaceutical/drug delivery arts to deliverypolynucleotides, proteins, small molecules, peptides, antigen, drugs,etc. to a patient, tissue, organ, cell, etc. The present invention alsoprovides for the preparation and screening of large collections ofpolymers for “hits” that are useful in the pharmaceutical and drugdelivery arts.

[0071] The β-amino ester polymers of the present invention provide forseveral different uses in the drug delivery art. The polymers with theirtertiary amine-containing backbones may be used to complexpolynucleotides and thereby enhance the delivery of polynucleotide andprevent their degradation. The polymers may also be used in theformation of nanoparticles or microparticles containing encapsulatedagents. Due to the polymers' properties of being biocompatible andbiodegradable, these formed particles are also biodegradable andbiocompatible and may be used to provide controlled, sustained releaseof the encapsulated agent. These particles may also be responsive to pHchanges given the fact that these polymers are typically notsubstantially soluble in aqueous solution at physiologic pH but are moresoluble at lower pH.

[0072] Polymers

[0073] The polymers of the present invention are poly(β-amino esters)containing tertiary amines in their backbones and salts thereof. Themolecular weights of the inventive polymers may range from 5,000 g/molto over 100,000 g/mol, more preferably from 4,000 g/mol to 50,000 g/mol.In a particularly preferred embodiment, the inventive polymers arerelatively non-cytotoxic. In another particularly preferred embodiment,the inventive polymers are biocompatible and biodegradable. In aparticularly preferred embodiment, the polymers of the present inventionhave pK_(a)s in the range of 5.5 to 7.5, more preferably between 6.0 and7.0. In another particularly preferred embodiment, the polymer may bedesigned to have a desired pK_(a) between 3.0 and 9.0, more preferablybetween 5.0 and 8.0. The inventive polymers are particularly attractivefor drug delivery for several reasons: 1) they contain amino groups forinteracting with DNA and other negatively charged agents, for bufferingthe pH, for causing endosomolysis, etc.; 2) they contain degradablepolyester linkages; 3) they can be synthesized from commerciallyavailable starting materials; and 4) they are pH responsive and futuregenerations could be engineered with a desired pK_(a). In screening fortransfection efficiency, the best performing polymers were hydrophobicor the diacrylate monomers were hydrophobic, and many had mono- ordi-hydroxyl side chains and/or linear, bis(secondary amines) as part oftheir structure.

[0074] The polymers of the present invention can generally be defined bythe formula (I):

[0075] The linkers A and B are each a chain of atoms covalently linkingthe amino groups and ester groups, respectively. These linkers maycontain carbon atoms or heteroatoms (e.g., nitrogen, oxygen, sulfur,etc.). Typically, these linkers are 1 to 30 atoms long, more preferably1-15 atoms long. The linkers may be substituted with varioussubstituents including, but not limited to, hydrogen atoms, alkyl,alkenyl, alkynl, amino, alkylamino, dialkylamino, trialkylamino,hydroxyl, alkoxy, halogen, aryl, heterocyclic, aromatic heterocyclic,cyano, amide, carbamoyl, carboxylic acid, ester, thioether,alkylthioether, thiol, and ureido groups. As would be appreciated by oneof skill in this art, each of these groups may in turn be substituted.The groups R₁, R₂, R₃, R₄, R₅, R₆, R₇, and R₈ may be any chemical groupsincluding, but not limited to, hydrogen atoms, alkyl, alkenyl, alkynl,amino, alkylamino, dialkylamino, trialkylamino, hydroxyl, alkoxy,halogen, aryl, heterocyclic, aromatic heterocyclic, cyano, amide,carbamoyl, carboxylic acid, ester, alkylthioether, thiol, and ureidogroups. In the inventive polymers, n is an integer ranging from 5 to10,000, more preferably from 10 to 500.

[0076] In a particularly preferred embodiment, the bis(secondary amine)is a cyclic structure, and the polymer is generally represented by theformula II:

[0077] In this embodiment, R₁ and R₂ are directly linked together asshown in formula II. Examples of inventive polymers in this embodimentinclude, but are not limited to formulas III and IV:

[0078] As described above in the preceding paragraph, any chemical groupthat satisfies the valency of each atom may be substituted for anyhydrogen atom.

[0079] In another particularly preferred embodiment, the groups R₁and/or R₂ are covalently bonded to linker A to form one or two cyclicstructures. The polymers of the present embodiment are generallyrepresented by the formula V in which both R₁ and R₂ are bonded tolinker A to form two cyclic structures:

[0080] The cyclic structures may be 3-, 4-, 5-, 6-, 7-, or 8-memberedrings or larger. The rings may contain heteroatoms and be unsaturated.Examples of polymers of formula V include formulas VI, VII, and VIII:

[0081] As described above, any chemical group that satisfies the valencyof each atom in the molecule may be substituted for any hydrogen atom.

[0082] In another embodiment, the polymers of the present invention cangenerally be defined by the formula (IX):

[0083] The linker B is a chain of atoms covalently linking the estergroups. The linker may contain carbon atoms or heteroatoms (e.g.,nitrogen, oxygen, sulfur, etc.). Typically, the linker is 1 to 30 atomslong, preferably 1-15 atoms long, and more preferably 2-10 atoms long.In certain embodiments, the linker B is a substituted or unsubstituted,linear or branched alkyl chain, preferably with 3-10 carbon atoms, morepreferably with 3, 4, 5, 6, or 7 carbon atoms. In other embodiments, thelinker B is a substituted or unsubstituted, linear or branchedheteroaliphatic chain, preferably with 3-10 atoms, more preferably with3, 4, 5, 6, or 7 atoms. In certain embodiments, the linker B iscomprises of repeating units of oxygen and carbon atoms. The linker maybe substituted with various substituents including, but not limited to,hydrogen atoms, alkyl, alkenyl, alkynyl, amino, alkylamino,dialkylamino, trialkylamino, hydroxyl, alkoxy, halogen, aryl,heterocyclic, aromatic heterocyclic, cyano, amide, carbamoyl, carboxylicacid, ester, thioether, alkylthioether, thiol, acyl, acetyl, and ureidogroups. As would be appreciated by one of skill in this art, each ofthese groups may in turn be substituted. Each of R1, R3, R4, R5, R6, R7,and R8 may be independently any chemical group including, but notlimited to, hydrogen atom, alkyl, alkenyl, alkynyl, amino, alkylamino,dialkylamino, trialkylamino, hydroxyl, alkoxy, halogen, aryl,heterocyclic, aromatic heterocyclic, cyano, amide, carbamoyl, carboxylicacid, ester, alkylthioether, thiol, acyl, acetyl, and ureido groups. Incertain embodiments, R1 includes hydroxyl groups. In other embodiments,R1 includes amino, alkylamino, or dialkylamino groups. In the inventivepolymer, n is an integer ranging from 5 to 10,000, more preferably from10 to 500.

[0084] In certain embodiments, the polymers of the present invention aregenerally defined as follows:

[0085] wherein

[0086] X is selected from the group consiting of C₁-C₆ lower alkyl,C₁-C₆ lower alkoxy, halogen, OR and NR₂; more preferably, methyl, OH, orNH₂;

[0087] R is selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, cyclic, heterocyclic, aryl, and heteroaryl;

[0088] each R′ is independently selected from the group consisting ofhydrogen, C₁-C₆ lower alkyl, C₁-C₆ lower alkoxy, hydroxy, amino,alkylamino, dialkylamino, cyano, thiol, heteroaryl, aryl, phenyl,heterocyclic, carbocyclic, and halogen; preferably, R′ is hydrogen,methyl, ethyl, propyl, isopropyl, butyl, methoxy, ethoxy, propoxy,isopropoxy, hydroxyl, amino, fluoro, chloro, or bromo; more preferably,R′ is fluoro, hydrogen, or methyl;

[0089] n is an integer between 3 and 10,000;

[0090] x is an integer between 1 and 10; preferably, x is an integerbetween 2 and 6;

[0091] y is an integer between 1 and 10; preferably, x is an intergerbetween 2 and 6; and

[0092] derivatives and salts thereof.

[0093] In certain embodiments, the polymers of the present invention aregenerally defined as follows:

[0094] wherein

[0095] X is selected from the group consiting of C₁-C₆ lower alkyl,C₁-C₆ lower alkoxy, halogen, OR and NR₂; more preferably, methyl, OH, orNH₂;

[0096] R is selected from the group consisting of hydrogen, alkyl,alkenyl, alkynyl, cyclic, heterocyclic, aryl, and heteroaryl;

[0097] each R′ is independently selected from the group consisting ofhydrogen, C₁-C₆ lower alkyl, C₁-C₆ lower alkoxy, hydroxy, amino,alkylamino, dialkylamino, cyano, thiol, heteroaryl, aryl, phenyl,heterocyclic, carbocyclic, and halogen; preferably, R′ is hydrogen,methyl, ethyl, propyl, isopropyl, butyl, methoxy, ethoxy, propoxy,isopropoxy, hydroxyl, amino, fluoro, chloro, or bromo; more preferably,R′ is fluoro, hydrogen, or methyl;

[0098] n is an integer between 3 and 10,000;

[0099] x is an integer between 1 and 10; preferably, x is an integerbetween 2 and 6;

[0100] y is an integer between 1 and 10; preferably, y is an intergerbetween 2 and 6;

[0101] z is an interger between 1 and 10; preferably, z is an integerbetween 2 and 6; and

[0102] derivatives and salts thereof.

[0103] In another embodiment, the bis(acrylate ester) unit in theinventive polymer is chosen from the following group of bis(acrylateester) units (A′-G′):

[0104] In certain embodiments, the polymer comprises the bis(acrylateester) G′.

[0105] In another embodiment, the bis(acrylate ester) unit in theinventive polymer is chosen from the following group of bis(acrylateester) units (A-PP):

[0106] Particularly preferred bis(acrylate esters) in this group includeE, F, M, U, JJ, KK, LL, C, and D.

[0107] In another embodiment, the amine in the inventive polymer ischosen from the following group of amines (1′-20′):

[0108] In certain embodiments, the polymer comprises the amine 5′. Inother embodiments, the polymer comprises amine 14′.

[0109] In another embodiment, the amine in the inventive polymer ischosen from the following group of amines (1-94):

[0110] In certain embodiments, the polymers include amines 6, 17, 20,28, 32, 36, 60, 61, 86, 89, or 94.

[0111] Particular examples of the polymers of the present inventioninclude:

[0112] Other particularly useful poly(beta-amino ester)s include:

[0113] In a particularly preferred embodiment, one or both of thelinkers A and B are polyethylene polymers. In another particularlypreferred embodiment, one or both of the linkers A and B arepolyethylene glycol polymers. Other biocompatible, biodegradablepolymers may be used as one or both of the linkers A and B.

[0114] In certain preferred embodiments, the polymers of the presentinvention are amine-terminated. In other embodiments, the polymers ofthe present invention are acrylate-terminated.

[0115] In certain embodiments, the average molecular weight of thepolymers of the present invention range from 1,000 g/mol to 50,000g/mol, preferably from 2,000 g/mol to 40,000 g/mol, more preferably from5,000 g/mol to 20,000 g/mol, and even more preferably from 10,000 g/molto 17,000 g/mol. Since the polymers of the present invention areprepared by a step polymerization, a broad, statistical distribution ofchain lengths is typically obtained. In certain embodiments, thedistribution of molecular weights in a polymer sample is narrowed bypurification and isolation steps known in the art. In other embodiments,the polymer mixture may be a blend of polymers of different molecularweights.

[0116] In another particularly preferred embodiment, the polymer of thepresent invention is a co-polymer wherein one of the repeating units isa poly(β-amino ester) of the present invention. Other repeating units tobe used in the co-polymer include, but are not limited to, polyethylene,poly(glycolide-co-lactide) (PLGA), polyglycolic acid, polymethacrylate,etc.

[0117] Synthesis of Polymers

[0118] The inventive polymers may be prepared by any method known in theart. Preferably the polymers are prepared from commercially availablestarting materials. In another preferred embodiment, the polymers areprepared from easily and/or inexpensively prepared starting materials.

[0119] In a particularly preferred embodiment, the inventive polymer isprepared via the conjugate addition of bis(secondary amines) tobis(acrylate esters). This reaction scheme is shown below:

[0120] Bis(secondary amine) monomers that are useful in the presentinventive method include, but are not limited to,N,N′-dimethylethylenediamine, piperazine, 2-methylpiperazine,1,2-bis(N-ethylamino)ethylene, and 4,4′-trimethylenedipiperidine.Diacrylate monomers that are useful in the present invention include,but are not limited to, 1,4-butanediol diacrylate, 1,4-butanedioldimethacrylate, 1,2-ethanediol diacrylate, 1,6-hexanediol diacrylate,1,5-pentanediol diacrylate, 2,5-hexanediol diacrylate, and1,3-propanediol diacrylate. Each of the monomers is dissolved in anorganic solvent (e.g., THF, CH₂Cl₂, MeOH, EtOH, CHCl₃, hexanes, toluene,benzene, CCl₄, glyme, diethyl ether, DMSO, DMF, etc.), preferably DMSO.The resulting solutions are combined, and the reaction mixture is heatedto yield the desired polymer. In other embodiments, the reaction isperformed without the use of a solvent thereby obviating the need forremoving the solvent after the synthesis. The reaction mixture is thenheated to a temperature ranging from 30° C. to 200° C., preferably 40°C. to 150° C., more preferably 50° C. to 100° C. In a particularlypreferred embodiment, the reaction mixture is heated to approximately40° C., 50° C., 60° C., 70° C., 80° C., or 90° C. In anotherparticularly preferred embodiment, the reaction mixture is heated toapproximately 75° C. In another embodiment, the reaction mixture isheated to approximately 100° C. The polymerization reaction may also becatalyzed. The reaction time may range from hours to days depending onthe polymerization reaction and the reaction conditions. As would beappreciated by one of skill in the art, heating the reaction tends tospeed up the rate of reaction requiring a shorter reaction time. Aswould be appreciated by one of skill in this art, the molecular weightof the synthesized polymer may be determined by the reaction conditions(e.g., temperature, starting materials, concentration, catalyst,solvent, time of reaction, etc.) used in the synthesis.

[0121] In another particularly preferred embodiment, the inventivepolymers are prepared by the conjugate addition of a primary amine to abis(acrylate ester). The use of primary amines rather than bis(secondaryamines) allows for a much wider variety of commercially availablestarting materials. The reaction scheme using primary amines rather thansecondary amines is shown below:

[0122] Primary amines useful in this method include, but are not limitedto, methylamine, ethylamine, isopropylamine, aniline, substitutedanilines, and ethanolamine. The bis(acrylate esters) useful in thismethod include, but are not limited to, 1,4-butanediol diacrylate,1,4-butanediol dimethacrylate, 1,2-ethanediol diacrylate, 1,6-hexanedioldiacrylate, 1,5-pentanediol diacrylate, 2,5-hexanediol diacrylate, and1,3-propanediol diacrylate. Each of the monomers is dissolved in anorganic solvent (e.g., THF, DMSO, DMF, CH₂Cl₂, MeOH, EtOH, CHCl₃,hexanes, CCl₄, glyme, diethyl ether, etc.). Organic solvents arepreferred due to the susceptibility of polyesters to hydrolysis.Preferably the organic solvent used is relatively non-toxic in livingsystems. In certain embodiments, DMSO is used as the organic solventbecause it is relatively non-toxic and is frequently used as a solventin cell culture and in storing frozen stocks of cells. Other preferredsolvents include those miscible with water such as DMSO, ethanol, andmethanol. The resulting solutions of monomers which are preferably at aconcentration between 0.01 M and 5 M, between approximately 0.1 M and 2M, more preferably between 0.5 M and 2 M, and most preferably between1.3 M and 1.8 M, are combined, and the reaction mixture is heated toyield the desired polymer. In certain embodiments, the reaction is runwithout solvent. Running the polymerization without solvent may decreasethe amount of cyclization products resulting from intramolecularconjugate addition reactions. The polymerization may be run at atemperature ranging from 20° C. to 200° C., preferably from 40° C. to100° C., more preferably from 50° C. to 75° C., even more preferablyfrom 50° C. to 60° C. In a particularly preferred embodiment, thereaction mixture is maintained at 20° C. In another particularlypreferred embodiment, the reaction mixture is heated to approximately50° C. In some embodiments, the reaction mixture is heated toapproximately 56° C. In yet another particularly preferred embodiment,the reaction mixture is heated to approximately 75° C. The reactionmixute may also be cooled to approximately 0° C. The polymerizationreaction may also be catalyzed such as with an organometallic catalyste,acid, or base. In another preferred embodiment, one or more types ofamine monomers and/or diacrylate monomers may be used in thepolymerization reaction. For example, a combination of ethanolamine andethylamine may be used to prepare a polymer more hydrophilic than oneprepared using ethylamine alone, and also more hydrophobic than oneprepared using ethanolamine alone.

[0123] In preparing the polymers of the present invention, the monomersin the reaction mixture may be combined in different ratio to effectmolecular weight, yield, end-termination, etc. of the resulting polymer.The ratio of amine monomers to diacrylate monomers may range from 1.6 to0.4, preferably from 1.4 to 0.6, more preferably from 1.2 to 0.8, evenmore preferably from 1.1 to 0.9. In certain embodiments, the ratio ofamine monomers to diacrylate monomers is approximately 1.0. For example,combining the monomers at a ratio of 1:1 typically results in highermolecular weight polymers and higher overall yields. Also, providing anexcess of amine monomers (i.e., amine-to-acrylate ratio>1) results inamine-terminated chains while providing an excess of acrylate monomer(i.e., amine-to-acrylate ration<1) results in acrylate-terminatedchains.

[0124] The synthesized polymer may be purified by any technique known inthe art including, but not limited to, precipitation, crystallization,chromatography, etc. In a particularly preferred embodiment, the polymeris purified through repeated precipitations in organic solvent (e.g.,diethyl ether, hexane, etc.). In a particularly preferred embodiment,the polymer is isolated as a hydrochloride, phosphate, or acetate salt.The resulting polymer may also be used as is without furtherpurification and isolation; thereby making it advantageous to use asolvent compatible with the assays to be used in assessing the polymers.For example, the polymers may be prepared in a non-toxic solvent such asDMSO, and the resulting solution of polymer may then be used in cellculture assays involving transfecting a nucleic acid into a cell. Aswould be appreciated by one of skill in this art, the molecular weightof the synthesized polymer and the extent of cross-linking may bedetermined by the reaction conditions (e.g., temperature, startingmaterials, concentration, order of addition, solvent, etc.) used in thesynthesis (Odian Principles of Polymerization 3rd Ed., New York: JohnWiley & Sons, 1991; Stevens Polymer Chemistry: An Introduction 2nd Ed.,New York: Oxford University Press, 1990; each of which is incorporatedherein by reference). The extent of cross-linking of the preparedpolymer may be minimized to between 1-20%, preferably between 1-10%,more preferably between 1-5%, and most preferably less than 2%. As wouldbe appreciated by those of skill in this art, amines or bis(acrylateester)s with nucleophlic groups are more susceptible to cross-linking,and measures may need to be taken to reduce cross-linking such aslowering the temperature or changing the concetration of the startingmaterials in the reaction mixture. Acrylates and other moieties withunsaturation or halogens are also susceptible to radical polymerizationwhich can lead to cross-linking. The extent of radical polymerizationand cross-linking may be reduced by reducing the temperature of thereaction mixture or by other means known in the art.

[0125] In one embodiment, a library of different polymers is prepared inparallel. The synthesis of a library of polymers may be carried outusing any of the teachings known in the art or described hereinregarding the synthesis of polymers of the invention. In one embodiment,a different amine and/or bis(acrylate ester) at a particularamine-to-acrylate ratio is added to each vial in a set of vials used toprepare the library or to each well in a multi-well plate (e.g., 96-wellplate). In one embodiment, the monomers are diluted to between 0.1 M and5 M, more preferably 0.5 M to 2 M, and most preferably at approximately1.6 M, in an organic solvent such as DMSO. The monomers may be combinedin different ratio to effect molecular weight, yield, end-termination,etc. For example, combining the monomers at a ratio of 1:1 typicallyyields higher molecular weight polymers and higher overall yields.Providing an excess of amine monomer results in amine-terminated chainswhile providing an excess of acrylate monomer results inacrylate-terminated chains. In some embodiments, no solvent is used inthe syntheis of the polymer. The array of vials or multi-well plate isincubated at a temperature and length of time sufficient to allowpolymerization of the monomers to occur as described herein. In certainpreferred embodiments, the time and temperature are chosen to effectnear complete incorporation (e.g., 50% conversion, 75% conversion, 80%conversion, 90% conversion, 95% conversion, >95% conversion, 98%conversion, 99% conversion, or >99% conversion) of all the monomer intopolymer. The polymerization reaction may be run at any temperaturesranging from 0° C. to 200° C. In one embodiment, the reaction mixturesare incubated at approximately 45° C. for approximately 5 days. Inanother embodiment, the reaction mixtures are incubated at approximately56° C. for approximately 5 days. In aother embodiment, the reactionmixtures are incubated at approximately 100° C. for approximately 5hours. The polymers may then be isolated and purified using techniquesknown in the art, or the resulting solutions of polymers may be usedwithout further isolation or purification. In certain embodiments, over1000 different polymers are prepared in parallel. In other embodiments,over 2000 different polymers are prepared in parallel. In still otherembodiments, over 3000 different polymers are prepared in parallel. Thepolymers may then be screened using high-throughput techniques toidentify polymers with a desired characteristic (e.g., solubility inwater, solubility at different pH's, solubility in various organicsolvents, ability to bind polynucleotides, ability to bind heparin,ability to bind small molecules, ability to form microparticles, abilityto increase tranfection efficiency, etc.). The polymers of the inventionmay be screened or used after synthesis without further precipitation,purification, or isolation of the polymer. The use of a non-toxicsolvent such as DMSO in the synthesis of the polymers allows for theeasy handling and use of the polymers after the synthesis of thepolymer. For instance, the solution of polymer in DMSO may be added to acell culture or other living system without a toxic effect on the cells.In certain embodiments the polymers may be screened for properties orcharacteristics useful in gene therapy (e.g., ability to bindpolynucleotides, increase in transfection efficiency, etc.). In otherembodiments the polymers may be screened for properties orcharacteristics useful in the art of tissue engineering (e.g., abilityto support tissue or cell growth, ability to promote cell attachment).In certain embodiments, the polymers are synthesized and assayed usingsemi-automated techniques and/or robotic fluid handling systems.

[0126] Polynucleotide Complexes

[0127] The ability of cationic compounds to interact with negativelycharged polynucleotides through electrostatic interactions is wellknown. Cationic polymers such as poly(lysine) have been prepared andstudied for their ability to complex polynucleotides. However, polymersstudied to date have incorporated amines at the terminal ends of short,conformationally flexible side chains (e.g., poly(lysine)) or accessibleamines on the surface of spherical or globular polyamines (e.g., PEI andPAMAM dendrimers). The interaction of the polymer with thepolynucleotide is thought to at least partially prevent the degradationof the polynucleotide. By neutralizing the charge on the backbone of thepolynucleotide, the neutral or slightly-positively-charged complex isalso able to more easily pass through the hydrophobic membranes (e.g.,cytoplasmic, lysosomal, endosomal, nuclear) of the cell. In aparticularly preferred embodiment, the complex is slightly positivelycharged. In another particularly preferred embodiment, the complex has apostive ζ-potential, more preferably the ζ-potential is between +1 and+30. In certain embodiments, agents such as polyacrylic acid (pAA), polyaspartic acid, polyglutamic acid, or poly-maleic acid may be used toprevent the serum inhibition of the polynucleotide/polymer complexes incultured cells in media with serum (Trubetskoy et al. “Rechargingcationic DNA complexes with highly charged polyanions for in vitro andin vivo gene delivery” Gene Therapy 10:261-271, 2003; incorporatedherein by reference).

[0128] The poly(β-amino esters) of the present invention possesstertiary amines in the backbone of the polymer. Although these aminesare more hindered, they are available to interact with a polynucleotide.Polynucleotides or derivatives thereof are contacted with the inventivepolymers under conditions suitable to form polynucleotide/polymercomplexes. In certain embodiments, the ratio of nitrogen in the polymer(N) to phosphate in the polynucleotide is 10:1, 15:1, 20:1, 25:1, 30:1,35:1, 40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1,95:1, 100:1, 110: 1, or 120:1. In certain embodiments, thepolymer-to-DNA (w/w) ratio is 10: 1, 15:1, 20: 1, 25:1, 30:1, 35:1,40:1, 45:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1,100:1, 110:1, 120:1, 130:1, 140:1, 150:1, or 200:1. The polymer ispreferably at least partially protonated so as to form a complex withthe negatively charged polynucleotide. In a preferred embodiment, thepolynucleotide/polymer complexes form nanoparticles that are useful inthe delivery of polynucleotides to cells. In a particularly preferredembodiment, the diameter of the nanoparticles ranges from 50-500 nm,more preferably the diameter of the nanoparticles ranges from 50-200 nm,and most preferably from 90-150 nm. The nanoparticles may be associatedwith a targeting agent as described below.

[0129] In certain embodiments, other agents may be added to thepolynucleotide:poly(betamino ester) complexes. In certain embodiments, aco-complexing agent is used. Co-complexing agents are known to bindpolynucleotides and/or increase transfection efficiency. Co-complexingagents usually have a high nitrogen density. Polylysine (PLL) andpolyethylenimine (PEI) are two examples of polymeric co-complexingagents. PLL has a molecular weight to nitrogen atom ratio of 65, and PEIhas a molecular weight to nitrogen atom ratio of 43. Any polymer with amolecular weight to nitrogen atom ratio in the range of 10-100,preferably 25-75, more preferably 40-70, may be useful as aco-complexing agent. The inclusion of a co-complexing agent in a complexmay allow one to reduce the amount of poly(beta-amino ester) in thecomplex. This becomes particularly important if the poly(beta-aminoester) is cytotoxic at higher concentrations. In the resulting complexeswith co-complexing agents, the co-complexing agent to polynucleotide(w/w) ratio may range from 0 to 2.0, preferably from 0.1 to 1.2, morepreferably from 0.1 to 0.6, and even more preferably from 0.1 to 0.4.

[0130] The transfection properties of various complexes of the inventionmay be determined by in vitro transfection studies (e.g., GFPtransfection in cultured cells) or in animal models. In certainembodiments, the complex used for transfection is optimized for aparticular cell type, polynucleotide to be delivered, poly(beta-aminoester), co-complexing agent (if one is used), disease process, method ofadministration (e.g., inhalation, oral, parenteral, IV, intrathecal,etc.), dosage regimen, etc.

[0131] Polynucleotide

[0132] The polynucleotide to be complexed or encapsulated by theinventive polymers may be any nucleic acid including but not limited toRNA and DNA. The polynucleotides may be of any size or sequence, andthey may be single- or double-stranded. In certain preferredembodiments, the polynucleotide is greater than 100 base pairs long. Incertain other preferred embodiments, the polynucleotide is greater than1000 base pairs long and may be greater than 10,000 base pairs long. Thepolynucleotide is preferably purified and substantially pure.Preferably, the polynucleotide is greater than 50% pure, more preferablygreater than 75% pure, and most preferably greater than 95% pure. Thepolynucleotide may be provided by any means known in the art. In certainpreferred embodiments, the polynucleotide has been engineered usingrecombinant techniques (for a more detailed description of thesetechniques, please see Ausubel et al. Current Protocols in MolecularBiology (John Wiley & Sons, Inc., New York, 1999); Molecular Cloning: ALaboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch, and Maniatis (ColdSpring Harbor Laboratory Press: 1989); each of which is incorporatedherein by reference). The polynucleotide may also be obtained fromnatural sources and purified from contaminating components foundnormally in nature. The polynucleotide may also be chemicallysynthesized in a laboratory. In a preferred embodiment, thepolynucleotide is synthesized using standard solid phase chemistry.

[0133] The polynucleotide may be modified by chemical or biologicalmeans. In certain preferred embodiments, these modifications lead toincreased stability of the polynucleotide. Modifications includemethylation, phosphorylation, end-capping, etc.

[0134] Derivatives of polynucleotides may also be used in the presentinvention. These derivatives include modifications in the bases, sugars,and/or phosphate linkages of the polynucleotide. Modified bases include,but are not limited to, those found in the following nucleoside analogs:2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C5-bromouridine, C5-fluorouridine,C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine,C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine,8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine. Modified sugarsinclude, but are not limited to, 2′-fluororibose, ribose,2′-deoxyribose, 3′-azido-2′,3′-dideoxyribose, 2′,3′-dideoxyribose,arabinose (the 2′-epimer of ribose), acyclic sugars, and hexoses. Thenucleosides may be strung together by linkages other than thephosphodiester linkage found in naturally occurring DNA and RNA.Modified linkages include, but are not limited to, phosphorothioate and5′-N-phosphoramidite linkages. Combinations of the various modificationsmay be used in a single polynucleotide. These modified polynucleotidesmay be provided by any means known in the art; however, as will beappreciated by those of skill in this art, the modified polynucleotidesare preferably prepared using synthetic chemistry in vitro.

[0135] The polynucleotides to be delivered may be in any form. Forexample, the polynucleotide may be a circular plasmid, a linearizedplasmid, a cosmid, a viral genome, a modified viral genome, anartificial chromosome, etc.

[0136] The polynucleotide may be of any sequence. In certain preferredembodiments, the polynucleotide encodes a protein or peptide. Theencoded proteins may be enzymes, structural proteins, receptors, solublereceptors, ion channels, pharmaceutically active proteins, cytokines,interleukins, antibodies, antibody fragments, antigens, coagulationfactors, albumin, growth factors, hormones, insulin, etc. Thepolynucleotide may also comprise regulatory regions to control theexpression of a gene. These regulatory regions may include, but are notlimited to, promoters, enhancer elements, repressor elements, TATA box,ribosomal binding sites, stop site for transcription, etc. In otherparticularly preferred embodiments, the polynucleotide is not intendedto encode a protein. For example, the polynucleotide may be used to fixan error in the genome of the cell being transfected.

[0137] The polynucleotide may also be provided as an antisense agent orRNA interference (RNAi) (Fire et al. Nature 391:806-811, 1998;incorporated herein by reference). Antisense therapy is meant toinclude, e.g., administration or in situ provision of single- ordouble-stranded oligonucleotides or their derivatives which specificallyhybridize, e.g., bind, under cellular conditions, with cellular mRNAand/or genomic DNA, or mutants thereof, so as to inhibit expression ofthe encoded protein, e.g., by inhibiting transcription and/ortranslation (Crooke “Molecular mechanisms of action of antisense drugs”Biochim. Biophys. Acta 1489(1):31-44, 1999; Crooke “Evaluating themechanism of action of antiproliferative antisense drugs” AntisenseNucleic Acid Drug Dev. 10(2):123-126, discussion 127, 2000; Methods inEnzymology volumes 313-314, 1999; each of which is incorporated hereinby reference). The binding may be by conventional base paircomplementarity, or, for example, in the case of binding to DNAduplexes, through specific interactions in the major groove of thedouble helix (i.e., triple helix formation) (Chan et al. J. Mol. Med.75(4):267-282, 1997; incorporated herein by reference).

[0138] In a particularly preferred embodiment, the polynucleotide to bedelivered comprises a sequence encoding an antigenic peptide or protein.Nanoparticles containing these polynucleotides can be delivered to anindividual to induce an immunologic response sufficient to decrease thechance of a subsequent infection and/or lessen the symptoms associatedwith such an infection. The polynucleotide of these vaccines may becombined with interleukins, interferon, cytokines, and adjuvants such ascholera toxin, alum, Freund's adjuvant, etc. A large number of adjuvantcompounds are known; a useful compendium of many such compounds isprepared by the National Institutes of Health and can be found on theWorld Wide Web(http:/www.niaid.nih.gov/daids/vaccine/pdf/compendium.pdf, incorporatedherein by reference; see also Allison Dev. Biol. Stand. 92:3-11, 1998;Unkeless et al. Annu. Rev. Immunol. 6:251-281, 1998; and Phillips et al.Vaccine 10:151-158,1992, each of which is incorporated herein byreference).

[0139] The antigenic protein or peptides encoded by the polynucleotidemay be derived from such bacterial organisms as Streptococccuspneumoniae, Haemophilus influenzae, Staphylococcus aureus, Streptococcuspyrogenes, Corynebacterium diphtheriae, Listeria monocytogenes, Bacillusanthracis, Clostridium tetani, Clostridium botulinum, Clostridiumperfringens, Neisseria meningitidis, Neisseria gonorrhoeae,Streptococcus mutans, Pseudomonas aeruginosa, Salmonella typhi,Haemophilus parainfluenzae, Bordetella pertussis, Francisellatularensis, Yersinia pestis, Vibrio cholerae, Legionella pneumophila,Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum,Leptospirosis interrogans, Borrelia burgdorferi, Camphylobacter jejuni,and the like; from such viruses as smallpox, influenza A and B,respiratory syncytial virus, parainfluenza, measles, HIV,varicella-zoster, herpes simplex 1 and 2, cytomegalovirus, Epstein-Barrvirus, rotavirus, rhinovirus, adenovirus, papillomavirus, poliovirus,mumps, rabies, rubella, coxsackieviruses, equine encephalitis, Japaneseencephalitis, yellow fever, Rift Valley fever, hepatitis A, B, C, D, andE virus, and the like; and from such fungal, protozoan, and parasiticorganisms such as Cryptococcus neoformans, Histoplasma capsulatum,Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsiaricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci,Chlamydial trachomatis, Plasmodium falciparum, Trypanosoma brucei,Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis,Schistosoma mansoni, and the like.

[0140] Microparticles

[0141] The poly(β-amino esters) of the present invention may also beused to form drug delivery devices. The inventive polymers may be usedto encapsulate agents including polynucleotides, small molecules,proteins, peptides, metals, organometallic compounds, etc. The inventivepolymers have several properties that make them particularly suitable inthe preparation of drug delivery devices. These include 1) the abilityof the polymer to complex and “protect” labile agents; 2) the ability tobuffer the pH in the endosome; 3) the ability to act as a “protonsponge” and cause endosomolysis; and 4) the ability to neutralize thecharge on negatively charged agents. In a preferred embodiment, thepolymers are used to form microparticles containing the agent to bedelivered. In a particularly preferred embodiment, the diameter of themicroparticles ranges from between 500 nm to 50 micrometers, morepreferably from 1 micrometer to 20 micrometers, and most preferably from1 micrometer to 10 micrometers. In another particularly preferredembodiment, the microparticles range from 1-5 micrometers. Theencapsulating inventive polymer may be combined with other polymers(e.g., PEG, PLGA) to form the microspheres.

[0142] Methods of Preparing Microparticles

[0143] The inventive microparticles may be prepared using any methodknown in this art. These include, but are not limited to, spray drying,single and double emulsion solvent evaporation, solvent extraction,phase separation, simple and complex coacervation, and other methodswell known to those of ordinary skill in the art. Particularly preferredmethods of preparing the particles are the double emulsion process andspray drying. The conditions used in preparing the microparticles may bealtered to yield particles of a desired size or property (e.g.,hydrophobicity, hydrophilicity, external morphology, “stickiness”,shape, etc.). The method of preparing the particle and the conditions(e.g., solvent, temperature, concentration, air flow rate, etc.) usedmay also depend on the agent being encapsulated and/or the compositionof the polymer matrix.

[0144] Methods developed for making microparticles for delivery ofencapsulated agents are described in the literature (for example, pleasesee Doubrow, M., Ed., “Microcapsules and Nanoparticles in Medicine andPharmacy,” CRC Press, Boca Raton, 1992; Mathiowitz and Langer, JControlled Release 5:13-22, 1987; Mathiowitz et al. Reactive Polymers6:275-283, 1987; Mathiowitz et al. J. Appl. Polymer Sci. 35:755-774,1988; each of which is incorporated herein by reference).

[0145] If the particles prepared by any of the above methods have a sizerange outside of the desired range, the particles can be sized, forexample, using a sieve.

[0146] Agent

[0147] The agents to be delivered by the system of the present inventionmay be therapeutic, diagnostic, or prophylactic agents. Any chemicalcompound to be administered to an individual may be delivered using theinventive microparticles. The agent may be a small molecule,organometallic compound, nucleic acid, protein, peptide, polynucleotide,metal, an isotopically labeled chemical compound, drug, vaccine,immunological agent, etc.

[0148] In a preferred embodiment, the agents are organic compounds withpharmaceutical activity. In another embodiment of the invention, theagent is a clinically used drug. In a particularly preferred embodiment,the drug is an antibiotic, anti-viral agent, anesthetic, steroidalagent, anti-inflammatory agent, anti-neoplastic agent, antigen, vaccine,antibody, decongestant, antihypertensive, sedative, birth control agent,progestational agent, anti-cholinergic, analgesic, anti-depressant,anti-psychotic, β-adrenergic blocking agent, diuretic, cardiovascularactive agent, vasoactive agent, non-steroidal anti-inflammatory agent,nutritional agent, etc.

[0149] In a preferred embodiment of the present invention, the agent tobe delivered may be a mixture of agents. For example, a local anestheticmay be delivered in combination with an anti-inflammatory agent such asa steroid. Local anesthetics may also be administered with vasoactiveagents such as epinephrine. To give another example, an antibiotic maybe combined with an inhibitor of the enzyme commonly produced bybacteria to inactivate the antibiotic (e.g., penicillin and clavulanicacid).

[0150] Diagnostic agents include gases; metals; commercially availableimaging agents used in positron emissions tomography (PET), computerassisted tomography (CAT), single photon emission computerizedtomography, x-ray, fluoroscopy, and magnetic resonance imaging (MRI);and contrast agents. Examples of suitable materials for use as contrastagents in MRI include gadolinium chelates, as well as iron, magnesium,manganese, copper, and chromium. Examples of materials useful for CATand x-ray imaging include iodine-based materials.

[0151] Prophylactic agents include, but are not limited to, antibiotics,nutritional supplements, and vaccines. Vaccines may comprise isolatedproteins or peptides, inactivated organisms and viruses, dead organismsand viruses, genetically altered organisms or viruses, and cellextracts. Prophylactic agents may be combined with interleukins,interferon, cytokines, and adjuvants such as cholera toxin, alum,Freund's adjuvant, etc. Prophylactic agents include antigens of suchbacterial organisms as Streptococccus pneumoniae, Haemophilusinfluenzae, Staphylococcus aureus, Streptococcus pyrogenes,Corynebacterium diphtheriae, Listeria monocytogenes, Bacillus anthracis,Clostridium tetani, Clostridium botulinum, Clostridium perfringens,Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus mutans,Pseudomonas aeruginosa, Salmonella typhi, Haemophilus parainfluenzae,Bordetella pertussis, Francisella tularensis, Yersinia pestis, Vibriocholerae, Legionella pneumophila, Mycobacterium tuberculosis,Mycobacterium leprae, Treponemapallidum, Leptospirosis interrogans,Borrelia burgdorferi, Camphylobacter jejuni, and the like; antigens ofsuch viruses as smallpox, influenza A and B, respiratory syncytialvirus, parainfluenza, measles, HIV, varicella-zoster, herpes simplex 1and 2, cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus,adenovirus, papillomavirus, poliovirus, mumps, rabies, rubella,coxsackieviruses, equine encephalitis, Japanese encephalitis, yellowfever, Rift Valley fever, hepatitis A, B, C, D, and E virus, and thelike; antigens of fungal, protozoan, and parasitic organisms such asCryptococcus neoformans, Histoplasma capsulatum, Candida albicans,Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii,Rickettsia typhi, Mycoplasmapneumoniae, Chlamydialpsittaci, Chlamydialtrachomatis, Plasmodiumfalciparum, Trypanosoma brucei, Entamoebahistolytica, Toxoplasma gondii, Trichomonas vaginalis, Schistosomamansoni, and the like. These antigens may be in the form of whole killedorganisms, peptides, proteins, glycoproteins, carbohydrates, orcombinations thereof.

[0152] Targeting Agents

[0153] The inventive micro- and nanoparticles may be modified to includetargeting agents since it is often desirable to target a particularcell, collection of cells, or tissue. A variety of targeting agents thatdirect pharmaceutical compositions to particular cells are known in theart (see, for example, Cotten et al. Methods Enzym. 217:618, 1993;incorporated herein by reference). The targeting agents may be includedthroughout the particle or may be only on the surface. The targetingagent may be a protein, peptide, carbohydrate, glycoprotein, lipid,small molecule, etc. The targeting agent may be used to target specificcells or tissues or may be used to promote endocytosis or phagocytosisof the particle. Examples of targeting agents include, but are notlimited to, antibodies, fragments of antibodies, low-densitylipoproteins (LDLs), transferrin, asialycoproteins, gp120 envelopeprotein of the human immunodeficiency virus (HIV), carbohydrates,receptor ligands, sialic acid, etc. If the targeting agent is includedthroughout the particle, the targeting agent may be included in themixture that is used to form the particles. If the targeting agent isonly on the surface, the targeting agent may be associated with (i.e.,by covalent, hydrophobic, hydrogen boding, van der Waals, or otherinteractions) the formed particles using standard chemical techniques.

[0154] Pharmaceutical Compositions

[0155] Once the microparticles or nanoparticles (polymer complexed withpolynucleotide) have been prepared, they may be combined with one ormore pharmaceutical excipients to form a pharmaceutical composition thatis suitable to administer to animals including humans. As would beappreciated by one of skill in this art, the excipients may be chosenbased on the route of administration as described below, the agent beingdelivered, time course of delivery of the agent, etc.

[0156] Pharmaceutical compositions of the present invention and for usein accordance with the present invention may include a pharmaceuticallyacceptable excipient or carrier. As used herein, the term“pharmaceutically acceptable carrier” means a non-toxic, inert solid,semi-solid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. Some examples of materials which canserve as pharmaceutically acceptable carriers are sugars such aslactose, glucose, and sucrose; starches such as corn starch and potatostarch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth;malt; gelatin; talc; excipients such as cocoa butter and suppositorywaxes; oils such as peanut oil, cottonseed oil; safflower oil; sesameoil; olive oil; corn oil and soybean oil; glycols such as propyleneglycol; esters such as ethyl oleate and ethyl laurate; agar; detergentssuch as Tween 80; buffering agents such as magnesium hydroxide andaluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline;Ringer's solution; ethyl alcohol; and phosphate buffer solutions, aswell as other non-toxic compatible lubricants such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releasingagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator. The pharmaceuticalcompositions of this invention can be administered to humans and/or toanimals, orally, rectally, parenterally, intracistemally,intravaginally, intranasally, intraperitoneally, topically (as bypowders, creams, ointments, or drops), bucally, or as an oral or nasalspray.

[0157] Liquid dosage forms for oral administration includepharmaceutically acceptable emulsions, microemulsions, solutions,suspensions, syrups, and elixirs. In addition to the active ingredients(i.e., microparticles, nanoparticles, polynucleotide/polymer complexes),the liquid dosage forms may contain inert diluents commonly used in theart such as, for example, water or other solvents, solubilizing agentsand emulsifiers such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, dimethylformamide, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fattyacid esters of sorbitan, and mixtures thereof. Besides inert diluents,the oral compositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

[0158] Injectable preparations, for example, sterile injectable aqueousor oleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension, or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables. Ina particularly preferred embodiment, the particles are suspended in acarrier fluid comprising 1% (w/v) sodium carboxymethyl cellulose and0.1% (v/v) Tween 80.

[0159] The injectable formulations can be sterilized, for example, byfiltration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

[0160] Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the particles withsuitable non-irritating excipients or carriers such as cocoa butter,polyethylene glycol, or a suppository wax which are solid at ambienttemperature but liquid at body temperature and therefore melt in therectum or vaginal cavity and release the microparticles.

[0161] Solid dosage forms for oral administration include capsules,tablets, pills, powders, and granules. In such solid dosage forms, theparticles are mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets, and pills, thedosage form may also comprise buffering agents.

[0162] Solid compositions of a similar type may also be employed asfillers in soft and hard-filled gelatin capsules using such excipientsas lactose or milk sugar as well as high molecular weight polyethyleneglycols and the like.

[0163] The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions which can beused include polymeric substances and waxes.

[0164] Solid compositions of a similar type may also be employed asfillers in soft and hard-filled gelatin capsules using such excipientsas lactose or milk sugar as well as high molecular weight polyethyleneglycols and the like.

[0165] Dosage forms for topical or transdermal administration of aninventive pharmaceutical composition include ointments, pastes, creams,lotions, gels, powders, solutions, sprays, inhalants, or patches. Theparticles are admixed under sterile conditions with a pharmaceuticallyacceptable carrier and any needed preservatives or buffers as may berequired. Ophthalmic formulation, ear drops, and eye drops are alsocontemplated as being within the scope of this invention.

[0166] The ointments, pastes, creams, and gels may contain, in additionto the particles of this invention, excipients such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc, and zinc oxide, or mixtures thereof.

[0167] Powders and sprays can contain, in addition to the particles ofthis invention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates, and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants suchas chlorofluorohydrocarbons.

[0168] Transdermal patches have the added advantage of providingcontrolled delivery of a compound to the body. Such dosage forms can bemade by dissolving or dispensing the microparticles or nanoparticles ina proper medium. Absorption enhancers can also be used to increase theflux of the compound across the skin. The rate can be controlled byeither providing a rate controlling membrane or by dispersing theparticles in a polymer matrix or gel.

[0169] These and other aspects of the present invention will be furtherappreciated upon consideration of the following Examples, which areintended to illustrate certain particular embodiments of the inventionbut are not intended to limit its scope, as defined by the claims.

EXAMPLES Example 1 Degradable Poly(β-Amino Esters): Synthesis,Characterization, and Self-Assembly with Plasmid DNA

[0170] Experimental Section

[0171] General Considerations. All manipulations involving live cells orsterile materials were performed in a laminar flow using standardsterile technique. ¹H NMR (300.100 MHz) and ¹³C NMR (75.467 MHz) spectrawere recorded on a Varian Mercury spectrometer. All chemical shiftvalues are given in ppm and are referenced with respect to residualproton or carbon signal from solvent. Organic phase gel permeationchromatography (GPC) was performed using a Hewlett Packard 1100 Seriesisocratic pump, a Rheodyneu Model 7125 injector with a 100-μL injectionloop, and two PL-Gel mixed-D columns in series (5 μm, 300×7.5 mm,Polymer Laboratories, Amherst, Mass.). THF/0.1 M piperidine was used asthe eluent at a flow rate of 1.0 mL/min. Data was collected using anOptilab DSP interferometric refractometer (Wyatt Technology, SantaBarbara, Calif.) and processed using the TriSEC GPC software package(Viscotek Corporation, Houston, Tex.). The molecular weights andpolydispersities of the polymers are reported relative to monodispersedpolystyrene standards. Aqueous phase GPC was performed by AmericanPolymer Standards (Mentor, Ohio) using Ultrahydrogel L and 120A columnsin series (Waters Corporation, Milford, Mass.). Water (1% acetic acid,0.3 M NaCl) was used as the eluent at a flow rate of 1.0 mL/min. Datawas collected using a Knauer differential refractometer and processedusing an IBM/PC GPC-PRO3.13 software package (Viscotek Corporation,Houston, Tex.). The molecular weights and polydispersities of thepolymers are reported relative to poly(2-vinylpyridine) standards. Forcytotoxicity assays, absorbance was measured using a DynatechLaboratories MR5000 microplate reader at 560 nmn.

[0172] Materials. N,N′-dimethylethylenediamine, piperazine, and4,4′-trimethylenedipiperidine were purchased from Aldrich ChemicalCompany (Milwaukee, Wis.). 1,4-butanediol diacrylate was purchased fromAlfa Aesar Organics (Ward Hill, Mass.).(3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide (MTT) waspurchased from Sigma Chemical Company (St. Louis, Mo.). Plasmid DNA(pCMV-Luc) was produced in E. coli (DH5α, a kind gift from Zycos, Inc.,Cambridge, Mass.), isolated with a Qiagen kit, and purified by ethanolprecipitation. NIH 3T3 cells were purchased from American Type CultureCollection (Manassas, Va.) and grown at 37° C., 5% CO₂ in Dulbecco'smodified Eagle's medium, 90%; fetal bovine serum, 10%; penicillin, 100units/mL; streptomycin, 100 μg/mL. All other materials and solvents wereused as received without further purification.

[0173] General Polymerization Procedure. In a typical experiment,1,4-butanediol diacrylate (0.750 g, 0.714 mL, 3.78 mmol) and diamine(3.78 mmol) were weighed into two separate vials and dissolved in THF (5mL). The solution containing the diamine was added to the diacrylatesolution via pipette. A Teflon-coated stirbar was added, the vial wassealed with a Teflon-lined screw-cap, and the reaction was heated at 50°C. After 48 hours, the reaction was cooled to room temperature anddripped slowly into vigorously stirring diethyl ether or hexanes.Polymer was collected and dried under vacuum prior to analysis.

[0174] Synthesis of Polymer 1. Polymer 1 was prepared according to thegeneral procedure outlined above. ¹H NMR δ (CDCl₃, 300 MHz) 4.11 (br t,4H), 2.75 (br t, J=7.05 Hz, 4H), 2.53 (br s, 4H), 2.50 (br t, (obsc),J=7.20 Hz, 4H), 2.28 (br s, 6H), 1.71, (br m, 4H). ¹³C NMR δ (CDCl₃,75.47 MHz) 172.55, 64.14, 55.31, 53.39, 42.47, 32.54, 25.53.

[0175] Synthesis of Polymer 2. Polymer 2 was prepared according to thegeneral procedure outlined above. ¹H NMR δ (CDCl₃, 300 MHz) 4.11 (br t,4H), 2.74 (br t, J=7.35, 4H), 2.56 (br m, 12H), 1.71 (br t, 4H). ¹³C NMRδ (CDCl₃, 75.47 MHz) 172.24, 64.19, 53.55, 52.75, 32.27, 25.52.

[0176] Synthesis of Polymer 3. Polymer 3 was prepared according to thegeneral procedure outlined above. ¹H NMR δ (CDCl₃, 300 MHz) 4.11 (br t,4H), 3.00 (br m, 4H), 2.79 (br m, 4H), 2.65 (br m, 4H), 2.11 (br m, 4H),1.70 (br m, 8H), 1.25 (br m, 12H). ¹³C NMR δ (CDCl₃, 75.47 MHz) 172.37,64.13, 53.89 (br), 36.74, 35.58, 32.11 (br), 25.45, 24.05.

[0177] Polymer Degradation Studies. The hydrochloride salts of polymers1-3 were dissolved in acetate buffer (1 M, pH=5.1) or HEPES buffer (1 M,pH=7.4) at a concentration of 5 mg/mL (the use of millimolarconcentrations of buffer resulted in substantial reduction of pH duringdegradation due to the production of acidic degradation products).Samples were incubated at 37° C. on a mechanical rotator, and aliquots(1 mL) were removed at appropriate time intervals. Aliquots were frozenimmediately in liquid nitrogen and lyophilized. Polymer was extractedfrom dried buffer salts using THF/0.1 M piperidine (1 mL), and sampleswere analyzed directly by GPC.

[0178] Formation of DNA/Polymer Complexes and Agarose Gel RetardationAssays. DNA/polymer complexes were formed by adding 50 μL of a plasmidDNA solution (pCMV Luc, 2 μg/50 μL in water) to a gently vortexingsolution of the hydrochloride salt of polymers 1-3 (50 μL in 25 mM MES,pH=6.0, concentrations adjusted to yield desired DNA/polymer weightratios). The samples were incubated at room temperature for 30 minutes,after which 20%L was run on a 1% agarose gel (HEPES, 20 mM, pH=7.2, 65V,30 min). Samples were loaded on the gel with a loading buffer consistingof 10% Ficoll 400 (Amersham Pharmacia Biotech, Uppsala, Sweden) in HEPES(25 mM, pH=7.2). Bromphenol blue was not included as a visual indicatorin the loading buffer, since this charged dye appeared to interfere withthe complexation of polymer and DNA. DNA bands were visualized under UVillumination by ethidium bromide staining.

[0179] Quasi-Elastic Laser Light Scattering (QELS) and Measurement ofζ-potentials. QELS experiments and ζ-potential measurements were madeusing a ZetaPALS dynamic light scattering detector (BrookhavenInstruments Corporation, Holtsville, N.Y., 15 mW laser, incidentbeam=676 nm). DNA/polymer complexes were formed as described above foragarose gel retardation assays. Samples were diluted with 900 μL ofHEPES (20 mM, pH=7.0), added to a gently vortexing sample of DNA/polymercomplex (total volume=1 mL, pH=7.0). Average particle sizes andζ-potentials were determined at 25° C. Correlation functions werecollected at a scattering angle of 90°, and particle sizes werecalculated using the MAS option of BIC's particle sizing software (ver.2.30) using the viscosity and refractive index of pure water at 25° C.Particle sizes are expressed as effective diameters assuming a lognormaldistribution. Average electrophoretic mobilities were measured at 25° C.using BIC PALS zeta potential analysis software and zeta potentials werecalculated using the Smoluchowsky model for aqueous suspensions. Threemeasurements were made on each sample, and the results are reported asaverage diameters and zeta potentials.

[0180] Cytotoxicity Assays. Immortalized NIH 3T3 cells were grown in96-well plates at an initial seeding density of 10,000 cells/well in 200μL growth medium (90% Dulbecco's modified Eagle's medium, 10% fetalbovine serum, penicillin 100 units/mL, streptomycin 100 μg/mL). Cellswere grown for 24 hours, after which the growth medium was removed andreplaced with 180 μL of serum-free medium. Appropriate amounts ofpolymer were added in 20 μL aliquots. Samples were incubated at 37° C.for 5 hours, and the metabolic activity of each well was determinedusing a MTT/thiazolyl blue assay: to each well was added 25 μL of a 5mg/mL solution of MTT stock solution in sterile PBS buffer. The sampleswere incubated at 37° C. for 2 hours, and 100 μL of extraction buffer(20% w/v SDS in DMF/water (1:1), pH=4.7) was added to each well. Sampleswere incubated at 37° C. for 24 hours. Optical absorbance was measuredat 560 nm with a microplate reader and expressed as a percent relativeto control cells.

[0181] Results and Discussion

[0182] Polymer Synthesis and Characterization

[0183] The synthesis of linear poly(amido amines) containing tertiaryamines in their backbones was reported by Ferruti et al. in 1970 via theaddition of bifunctional amines to bisacrylamides (Anderson Nature392(Suppl.):25-30, 1996; Friedman Nature Med. 2:144-147, 1996; CrystalScience 270:404-410, 1995; Mulligan Science 260:926-932, 1993; each ofwhich is incorporated herein by reference). Linear poly(amido amines)were initially investigated as heparin and ion complexing biomaterials(Ferruti et al. Advances in Polymer Science 58:55-92, 1984; Ferruti etal. Biomaterials 15:1235-1241, 1994; Ferruti et al. Macromol. Chem.Phys. 200:1644-1654, 1999; Ferruti et al. Biomaterials 15:1235-1241,1994; each of which is incorporated herein by reference). Dendriticpoly(amido amines) (PAMAMs) have seen increasing use in gene transferapplications due to their ability to complex DNA (Kukowska-Latallo etal. Proc. Natl. Acad. Sci. USA 93:4897-4902, 1996; Tang et al.Bioconjugate Chem. 7:703-714, 1996; Haensler et al. Bioconjugate Chem.4:372-379, 1993; each of which is incorporated herein by reference), anda recent report describes the application of a family of linearpoly(amido amines) to cell transfection and cytotoxicity studies (Hillet al. Biochim. Biophys. Acta 1427:161-174, 1999; incorporated herein byreference). In contrast, analogous poly(ester amines) formed from theMichael-type addition of bifunctional amines to diacrylate esters havereceived less attention (Danusso et al. Polymer 11:88-113, 1970; Ferrutiet al. Polymer 26:1336, 1985; Ferruti et al. Advances in Polymer Science58:55-92, 1984; Ferruti et al. Biomaterials 15:1235-1241, 1994; Ferrutiet al. Macromol. Chem. Phys. 200:1644-1654, 1999; Ferruti et al.Biomaterials 15:1235-1241, 1994; Kargina et al. Vysokomol. Soedin.Seriya A 28:1139-1144, 1986; Rao et al. J. Bioactive and CompatiblePolymers 14:54-63, 1999; each of which is incorporated herein byreference).

[0184] The poly(amino ester) approach presents a particularly attractivebasis for the development of new polymeric transfection vectors forseveral reasons: 1) the polymers contain the requisite amines andreadily degradable linkages, 2) multiple analogs could potentially besynthesized directly from commercially available starting materials, and3) if the resulting polymers were useful as DNA condensing agents,future generations of polymer could easily be engineered to possessamine pK_(a) values spanning the range of physiologically relevant pH.This last point was particularly intriguing, because the bufferingcapacity of polyamines has recently been implicated as a factorinfluencing the escape of DNA from cell endosomes following endocytosis(Boussif et al. Proc. Natl. Acad. Sci. USA 92:7297-7301, 1995; Haensleret al. Bioconjugate Chem. 4:372-379, 1993; Behr Chimia 51:34-36, 1997;Demeneix et al., in Artificial Self-Assembling Systems for Gene Delivery(Felgner et al., Eds.), American Chemical Society, Washington, D.C.,1996, pp. 146-151; Kabanov et al., in Self-Assembling Complexes for GeneDelivery: From Laboratory to Clinical Trial, John Wiley and Sons, NewYork, 1998; each of which is incorporated herein by reference). Whilethe complexation of DNA with a cationic polymer is required to compactand protect DNA during early events in the transfection process, DNA andpolymer must ultimately decomplex in the nucleus to allow efficienttranscription (Luo et al. Nat. Biotechnol. 18:33-37, 2000; incorporatedherein by reference). In view of this requirement, degradablepolycations could play an important role in “vector unpackaging” eventsin the nucleus (Luo et al. Nat. Biotechnol. 18:33-37, 2000; Schaffer etal. Biotechnol. Bioeng. 67:598-606, 2000; Kabanov Pharm. Sci. Technol.Today 2:365-372, 1999; each of which is incorporated herein byreference). Finally, we hypothesized that polymers of this generalstructure, and the β-amino acid derivatives into which they wouldpresumably degrade, would be significantly less toxic than poly(lysine)and PEI. As outlined above, degradable polycations (Putnam et al.Macromolecules 32:3658-3662, 1999; Lim et al. J. Am. Chem. Soc.121:5633-5639, 1999; Lim et al. J. Am. Chem. Soc. 122:6524-6525, 2000;each of which is incorporated herein by reference) and linear polymerscontaining relatively hindered amines located close to the polymerbackbone (Gonzalez et al. Bioconjugate Chem. 10:1068-1074, 1999;incorporated herein by reference) are less toxic than poly(lysine) andPEI.

[0185] The synthesis of polymers 1-3 via the addition of thebis(secondary amines), N,N′-dimethylethylenediamine, piperazine, and4,4′-trimethylenedipiperidine, to 1,4-butanediol diacrylate wasinvestigated (Danusso et al. Polymer 11:88-113, 1970; Kargina et al.Vysokomol. Soedin. Seriya A 28:1139-1144, 1986; each of which isincorporated herein by reference). The polymerization of these monomersproceeded in THF and CH₂Cl₂ at 50° C. to yield the correspondingpolymers in up to 86% yields (Table 1). Polymers were purified throughrepeated precipitation into diethyl ether or hexane. Polymer 1 wasisolated as a clear viscous liquid; polymers 2 and 3 were obtained aswhite solids after drying under high vacuum. Alternatively, polymers 1-3could be isolated as solid hydrochloride salts upon addition of diethylether/HCl to a solution of polymer in THF or CH₂Cl₂. All three polymerswere soluble in organic solvents such as THF, CH₂Cl₂, CHCl₃, and MeOHand were also soluble in water at reduced pH. Polymer 1 and thehydrochloride salts of polymers 1-3 were freely soluble in water.

[0186] The molecular weights of polymers 1-3 and their correspondinghydrochloride salts were determined by both organic and aqueous phasegel permeation chromatography (GPC). Polymer molecular weights (M_(n))ranged from up to 5,800 for polymer 1 to up to 32,000 for polymer 3,relative to polystyrene standards. Molecular weight distributions forthese polymers were monomodal with polydispersity indices (PDIs) rangingfrom 1.55 to 2.55. Representative molecular weight data are presented inTable 1. While the synthesis of linear poly(amido amines) is generallyperformed using alcohols or water as solvents (Danusso et al. Polymer11:88-113, 1970; Ferruti et al. Polymer 26:1336, 1985; Ferruti et al.Advances in Polymer Science 58:55-92, 1984; Ferruti et al. Biomaterials15:1235-1241, 1994; Ferruti et al. Macromol. Chem. Phys. 200:1644-1654,1999; Ferruti et al. Biomaterials 15:1235-1241, 1994; each of which isincorporated herein by reference), anhydrous THF and CH₂Cl₂ wereemployed in the synthesis of poly(β-amino esters) to minimize hydrolysisreactions during the synthesis. The yields and molecular weights ofpolymers synthesized employing CH₂Cl₂ as solvent were generally higherthan those of polymers synthesized in THF (Table 1) (Polymer 1 could notby synthesized in CH₂Cl₂. The color of the reaction solution progressedfrom colorless to an intense pink color almost immediately after theintroduction of a solution of N,N′-dimethylethylenediamine in CH₂Cl₂ toa solution of 1,4-butanediol diacrylate in CH₂Cl₂ (see ExperimentalSection above). The color progressed to light orange over the course ofthe reaction, and an orange polymer was isolated after precipitationinto hexane. The isolated polymer was insoluble in CH₂Cl₂, THF, andwater at reduced pH and was not structurally characterized. This problemwas not encountered for the analogous reaction in THF.). TABLE 1Representative Molecular Weight Data for Polymers 1-3. Polymer SolventM_(n) ^(c) PDI Yield, % 1^(a) THF — — —^(d) 1^(a) CH₂Cl₂ — — 82% 2^(a)THF 10.000 1.77 64% 2^(a) CH₂Cl₂ 17.500 2.15 75% 3^(a) THF 24.400 1.5558% 3^(a) CH₂Cl₂ 30.800 2.02 70% 1^(b) THF  5.800 2.83 55% 2^(b) CH₂Cl₂16.500 2.37 80%^(e) 3^(b) CH₂Cl₂ 31.200 2.55 86%^(e)

[0187] The structures of polymers 1-3 were confirmed by ¹H and ¹³C NMRspectroscopy. These data indicate that the polymers were formed throughthe conjugate addition of the secondary amines to the acrylate moietiesof 1,4-butanediol diacrylate and not through the formation of amidelinkages under our reaction conditions. Additionally, the newly formedtertiary amines in the polymer backbones do not participate insubsequent addition reactions with diacrylate monomer, which would leadto branching or polymer crosslinking. This fortunate result appears tobe unique to polymers of this type produced from bis(secondary amine)monomers. The synthesis of analogous polymers employing difunctionalprimary amines as monomers (such as 1,4-diaminobutane) may lead topolymer branching and the formation of insoluble crosslinked polymernetworks if conditions are not explicitly controlled.

[0188] In view of the juxtaposition of amines and esters within thebackbones of polymers 1-3, we were initially concerned that hydrolysismight occur too rapidly for the polymers to be of practical use. Forexample, poly(4-hydroxy-L-proline ester) andpoly[α-(4-aminobutyl)-L-glycolic acid] degrade quite rapidly nearneutral pH, having half lives of roughly 2 hr (Lim et al. J. Am. Chem.Soc. 121:5633-5639, 1999; incorporated herein by reference) and 30 min(Lim et al. J. Am. Chem. Soc. 122:6524-6525, 2000; incorporated hereinby reference), respectively (Such rapid degradation times did notpreclude the application of these polymers to gene delivery (Seereferences, Lim et al. J. Am. Chem. Soc. 121:5633-5639, 1999; Lim et al.J. Am. Chem. Soc. 122:6524-6525, 2000; each of which is incorporatedherein by reference). However, extremely rapid degradation ratesgenerally introduce additional concerns surrounding the manipulation,storage, and application of degradable polymers.). Analysis of polymers1 and 2 by aqueous GPC using 1% acetic acid/water as eluent, however,revealed that degradation was sufficiently slow in acidic media. Forexample, the GPC traces of polymers 1 and 2 sampled under theseconditions over a period of 4-5 hours revealed no changes in molecularweights or polydispersities (Polymer 3 could not be analyzed by aqueousGPC.). We were also concerned that significant backbone hydrolysis mightoccur during the isolation of the hydrochloride salts of polymers 1-3.To prevent hydrolysis during the protonation and isolation of thesepolymers, anhydrous solvents were employed and reactions were performedunder an argon atmosphere. Analysis of the polymers before and afterprotonation revealed no observable hydrolysis. For example, the GPCtrace of a sample of polymer 3 after precipitation from CH₂Cl₂ with 1.0M diethyl ether/HCl (M_(n)=15,300; PDI=1.90) was virtually identical tothe molecular weight of the polymer prior to protonation (M_(n)=15,700;PDI=1.92) and no lower molecular weight species were evident(Comparative GPC data were collected employing THF/0.1M piperidine aseluent (see Experimental Section above). The HCl salts of the polymerswere insoluble in THF, but were soluble in THF/0.1 M piperidineconcomitant with the production of a fine white precipitate which wasfiltered prior to injection.). Solid samples of polymers 1-3 could bestored for several months without detectable decreases in molecularweight.

[0189] Polymers 1-3 were specifically designed to degrade via hydrolysisof the ester bonds in the polymer backbones. However, an additionalconcern surrounding the overall stability and biocompatibility of thesepolymers is the potential for unwanted degradation to occur throughretro-Michael reaction under physiological conditions. Because thesepolymers were synthesized via the Michael-type reaction of a secondaryamine to an acrylate ester, it is possible that the polymers couldundergo retro-Michael reaction to regenerate free acrylate groups,particularly under acidic conditions. Acrylate esters are potentialDNA-alkylating agents and are therefore suspected carcinogens (forrecent examples, see: Schweikl et al. Mutat. Res. 438:P71-P78, 1999;Yang et al. Carcinogenesis 19:P 1117-P1125, 1998; each of which isincorporated herein by reference). Because these polymers are expectedto encounter the reduced pH environment within the endosomal vesicles ofcells (pH=5.0-5.5) during transfection, we addressed the potential forthe degradation of these polymers to occur through a retro-Michaelpathway.

[0190] Under extremely acidic (pH<3) or basic (pH>12) conditions,polymers 1-3 degraded rapidly and exclusively to 1,4-butanediol and theanticipated bis(β-amino acid) byproducts 4a-6a as determined by ¹H NMRspectroscopy. No spectroscopic evidence for retro-Michael addition underthese conditions was found. It is worth noting that bis(P-amino acid)degradation products 4a-6a would be less likely to undergo aretro-Michael reaction, as acrylic acids are generally less activatedMichael addition partners (Perlmutter, P., in Conjugate AdditionReactions in Organic Synthesis, Pergamon Press, New York, 1992;incorporated herein by reference). Further degradation of compounds4a-6a under these conditions was not observed.

[0191] The kinetics of polymer degradation were investigated under therange of conditions likely to be encountered by these polymers duringtransfection. Degradation was monitored at 37° C. at buffered pH valuesof 5.1 and 7.4 in order to approximate the pH of the environments withinendosomal vesicles and the cytoplasm, respectively. The hydrochloridesalts of polymers 1-3 were added to the appropriate buffer, incubated at37° C., and aliquots were removed at appropriate times. Aliquots werefrozen immediately, lyophilized, and polymer was extracted into THF/0.1M piperidine for analysis by GPC. FIG. 1 shows the degradation profilesof polymers 1-3 as a function of time. The polymers degraded more slowlyat pH 5.1 than at pH 7.4. Polymers 1-3 displayed similar degradationprofiles at pH 5.1, each polymer having a half-life of approximately 7-8hours. In contrast, polymers 1 and 3 were completely degraded in lessthan 5 hours at pH 7.4. These results are consistent with thepH-degradation profiles of other amine-containing polyesters, such aspoly(4-hydroxy-L-proline ester), in which pendant amine functionalitiesare hypothesized to act as intramolecular nucleophilic catalysts andcontribute to more rapid degradation at higher pH (Lim et al. J. Am.Chem. Soc. 121:5633-5639, 1999; Lim et al. J. Am. Chem. Soc.122:6524-6525, 2000; each of which is incorporated herein by reference).While the possibility of intramolecular assistance cannot be ruled out,it is less likely for polymers 1-3 because the tertiary amines in thesepolymers should be less nucleophilic. The degradation of polymer 2occurred more slowly at pH 7.4 than at pH 5.1 (FIG. 1). This anomalousbehavior is most likely due to the incomplete solubility of polymer 2 atpH 7.4 and the resulting heterogeneous nature of the degradation milieu(Polymers 2 and 3 are not completely soluble in water at pH 7.4. Whilepolymer 3 dissolved relatively rapidly during the degradationexperiment, solid particles of polymer 2 were visible for several days.

[0192] Cytotoxicity Assays

[0193] Poly(lysine) and PEI have been widely studied as DNA condensingagents and transfection vectors (Luo et al. Nat. Biotechnol. 18:33-37,2000; Behr Acc. Chem. Res. 26:274-278, 1993; Zauner et al. Adv. DrugDel. Rev. 30:97-113, 1998; Kabanov et al. Bioconjugate Chem. 6:7-20,1995; Boussif et al. Proc. Natl. Acad. Sci. USA 92:7297-7301, 1995; BehrChimia 51:34-36, 1997; Demeneix et al., in Artificial Self-AssemblingSystems for Gene Delivery (Felgner et al., Eds.), American ChemicalSociety, Washington, D.C., 1996, pp. 146-151; Kabanov et al., inSelf-Assembling Complexes for Gene Delivery: From Laboratory to ClinicalTrial, John Wiley and Sons, New York, 1998; each of which isincorporated herein by reference) and are the standards to which newpolymeric vectors are often compared (Putnam et al. Macromolecules32:3658-3662, 1999;Lim et al. J. Am. Chem. Soc. 121:5633-5639, 1999; Limet al. J. Am. Chem. Soc. 122:6524-6525, 2000; Gonzalez et al.Bioconjugate Chem. 10:1068-1074, 1999; each of which is incorporatedherein by reference). Unfortunately, as outlined above, these polymersare also associated with significant levels of cytotoxicity and highlevels of gene expression are usually realized only at a substantialcost to cell viability. To determine the toxicity profile of polymers1-3, a MTT/thiazolyl blue dye reduction assay using the NIH 3T3 cellline and the hydrochloride salts of polymers 1-3 was conducted as aninitial indicators. The 3T3 cell line is commonly employed as a firstlevel screening population for new transfection vectors, and the MTTassay is generally used as an initial indicator of cytotoxicity, as itdetermines the influences of added substances on cell growth andmetabolism (Hansen et al. Immunol. Methods 119:203-210, 1989;incorporated herein by reference).

[0194] Cells were incubated with polymer 1 (M_(n)=5 800), polymer 2(M_(n)=11 300), and polymer 3 (M_(n)=22 500) as described in theExperimental Section. As shown in FIG. 2, cells incubated with thesepolymers remained 100% viable relative to controls at concentrations ofpolymer up to 100 μg/mL. These results compare impressively to dataobtained for cell populations treated with PEI (M_(n)=25 000), includedas a positive control for our assay as well as to facilitate comparisonto this well-known transfection agent. Fewer than 30% of cells treatedwith PEI remained viable at a polymer concentration of 25 μg/mL, andcell viability was as low as 10% at higher concentrations of PEI underotherwise identical conditions. An analogous MTT assay was performedusing independently synthesized bis(β-amino acid)s 4a-6a to screen thecytotoxicity of the hydrolytic degradation products of these polymers.(Bis(β-amino acid)s 4a-6a should either be biologically inert or possessmild or acute toxicities which are lower than traditional polycationictransfection vectors. In either case, the degradation of these materialsshould facilitate rapid metabolic clearance.). Compounds 4a-6a and1,4butanediol did not perturb cell growth or metabolism in this initialscreening assay (data not shown). A more direct structure/function-basedcomparison between polymers 1-3 and PEI cannot be made due todifferences in polymer structure and molecular weight, both of whichcontribute to polycation toxicity. Nonetheless, the excellentcytotoxicity profiles of polymers 13 alone suggested that they wereinteresting candidates for further study as DNA condensing agents.

[0195] Self Assembly of Polymers 1-3 with Plasmid DNA

[0196] The tendency of cationic polyamines to interact electrostaticallywith the polyanionic backbone of DNA in aqueous solution is well known.Provided that the polymers are sufficiently protonated at physiologicalpH, and that the amines are sterically accessible, such interactions canresult in a self-assembly process in which the positively and negativelycharged polymers form well-defined conjugates (Kabanov et al., inSelf-Assembling Complexes for Gene Delivery: From Laboratory to ClinicalTrial, John Wiley and Sons, New York, 1998; each of which isincorporated herein by reference). The majority of polyaminesinvestigated as DNA-complexing agents and transfection vectors haveincorporated amines at the terminal ends of short, conformationallyflexible side chains (e.g., poly(lysine) and methacrylate/methacrylamidepolymers) (Zauner et al. Adv. Drug Del. Rev. 30:97-113, 1998; Kabanov etal. Bioconjugate Chem. 6:7-20, 1995; van de Wetering et al. BioconjugateChem. 10:589-597, 1999; each of which is incorporated herein byreference), or accessible amines on the surfaces of spherical orglobular polyamines (e.g., PEI and PAMAM dendrimers) (Boussif et al.Proc. Natl. Acad. Sci. USA 92:7297-7301, 1995; Kukowska-Latallo et al.Proc. Natl. Acad. Sci. USA 93:4897-4902, 1996; Tang et al. BioconjugateChem. 7:703-714, 1996; Haensler et al. Bioconjugate Chem. 4:372-379,1993; each of which is incorporated herein by reference). Becausepolymers 1-3 contain tertiary amines, and those tertiary amines arelocated in a sterically crowded environment (flanked on two sides by thepolymer backbones), we were initially concerned that the protonatedamines might not be sufficiently able to interact intimately with DNA.

[0197] The ability of polymers 1-3 to complex plasmid DNA wasdemonstrated through an agarose gel shift assay. Agarose gelelectrophoresis separates macromolecules on the basis of both charge andsize. Therefore, the immobilization of DNA on an agarose gel in thepresence of increasing concentrations of a polycation has been widelyused as an assay to determine the point at which complete DNA chargeneutralization is achieved (Putnam et al. Macromolecules 32:3658-3662,1999; Lim et al. J. Am. Chem. Soc. 121:5633-5639, 1999; Lim et al. J.Am. Chem. Soc. 122:6524-6525, 2000; Gonzalez et al. Bioconjugate Chem.10: 1068-1074, 1999; each of which is incorporated herein by reference).As mentioned above, the hydrochloride salts of polymers 1-3 are solublein water. However, polymers 2 and 3 are not completely soluble at pH 7.2over the full range of desired polymer concentrations. Therefore,DNA/polymer complexes were prepared in MES buffer (25 mM, pH=6.0).DNA/polymer complexes were prepared by adding an aqueous solution of DNAto vortexing solutions of polymer in MES at desired DNA/polymerconcentrations (see Experimental Section). The resulting DNA/polymercomplexes remained soluble upon dilution in the electrophoresis runningbuffer (20 mM HEPES, pH=7.2) and data were obtained at physiological pH.As a representative example, FIG. 3 depicts the migration of plasmid DNA(pCMV-Luc) on an agarose gel in the presence of increasingconcentrations of polymer 1.

[0198] As shown in FIG. 3, retardation of DNA migration begins at DNA/1ratios as low as 1:0.5 (w/w) and migration is completely retarded atDNA/polymer ratios above 1:1.0 (w/w) (DNA/polymer weight ratios ratherthan DNA/polymer charge ratios are reported here. Although bothconventions are used in the literature, we find weight ratios to be morepractical and universal, since the overall charge on a polyamine issubject to environmental variations in pH and temperature. WhileDNA/polymer charge ratios are descriptive for polymers such aspoly(lysine), they are less meaningful for polymers such as PEI and 1-3which incorporate less basic amines.). Polymers 2 and 3 completelyinhibit the migration of plasmid DNA at DNA/polymer ratios (w/w) above1:10 and 1:1.5, respectively (data not shown). These results varymarkedly from gel retardation experiments conducted using model“monomers.” Since the true monomers and the degradation products ofpolymers 1-3 do not adequately represent the repeat units of thepolymers, we used bis(methyl ester)s 4b-6b to examine the extent towhich the polyvalency and cooperative binding of polycations 1-3 isnecessary to achieve DNA immobilization. “Monomers” 4b-6b did notinhibit the migration of DNA at DNA/“monomer” ratios (w/w) of up to 1:30(data not shown).

[0199] The reasons for the less-efficient complexation employing polymer2 in the above gel electrophoresis assays most likely results fromdifferences in the pK_(a) values of the amines in these polymers. Thedirect measurement of the pK_(a) values of polymers 1-3 is complicatedby their degradability. However, we predict the range of pK_(a) valuesof the amines in polymers 1 and 2 to extend from approximately 4.5 and8.0 for polymer 1, to 3.0 and 7.0 for polymer 2, based on comparisons tostructurally related poly(β-amino amides) (The pK_(a) values ofstructurally-related poly(β-amino amides) containing piperazine anddimethylethylene diamine units in their backbones have been reported.Barbucci et al. Polymer 21:81-85, 1980; Barbucci et al. Polymer19:1329-1334, 1978; Barbucci et al. Macromolecules 14:1203-1209, 1981;each of which is incorporated herein by reference). As a result, polymer2 should be protonated to a lesser extent than polymer 1 atphysiological or near-neutral pH, and would therefore be a lesseffective DNA condensing agent. The range of pK_(a) values for polymer 3should be higher than the range for polymers 1 and 2 due to theincreased distance between the nitrogen atoms. Accordingly, polymer 3forms complexes with DNA at substantially reduced concentrationsrelative to polymer 2.

[0200] Agarose gel retardation assays are useful in determining theextent to which polycations interact with DNA. To be useful transfectionagents, however, polycations must also be able to self-assemble plasmidDNA into polymer/DNA complexes small enough to enter a cell throughendocytosis. For most cell types, this size requirement is on the orderof 200 nm or less (Zauner et al. Adv. Drug Del. Rev. 30:97-113, 1998;incorporated herein by reference), although larger particles can also beaccomodated (Demeneix et al., in Artificial Self-Assembling Systems forGene Delivery (Felgner et al., Eds.), American Chemical Society,Washington, D.C., 1996, pp. 146-151; Kabanov et al., in Self-AssemblingComplexes for Gene Delivery: From Laboratory to Clinical Trial, JohnWiley and Sons, New York, 1998; each of which is incorporated herein byreference). The ability of polymers 1-3 to compact plasmid DNA intonanometer-sized structures was determined by quasi-elastic laser lightscattering (QELS), and the relative surface charges of the resultingcomplexes were quantified through ζ-potential measurements. DNA/polymerparticles used for particle sizing and 1-potential measurements wereformed as described above for agarose gel electrophoresis assays anddiluted in 20 mM HEPES buffer (pH=7.0) for analysis, as described in theExperimental Section.

[0201] Polymer 1 formed complexes with diameters ranging from 90-150 nmat DNA/polymer ratios above 1:2 (w/w), and polymer 2 condensed DNA intoparticles on the order of 60-125 nm at DNA/polymer ratios above 1:10.These results are consistent with the data obtained from agarose gelelectrophoresis experiments above. However, the particles in theseexperiments aggregated over a period of hours to yield larger complexeswith diameters in the range of 1-2 microns. The tendency of theseparticles to aggregate can be rationalized by the low ζ-potentials ofthe DNA/polymer particles observed under these conditions. For example,complexes formed from polymer 1 at DNA/polymer ratios above 1:10 hadaverage ζ-potentials of +4.51 (±10.50) mV. The ζ-potentials of complexesformed from polymer 2 at DNA/polymer ratios above 1:20 were lower,reaching a limiting value of +1.04 (±10.57) mV. These differencescorrelate with the estimated pK_(a) values for these polymers, as thesurfaces of particles formed from polymer 1 would be expected toslightly more protonated than particles formed from polymer 2 at pH=7.0.

[0202] Polymer 3 formed complexes with diameters in the range of 50-150nm at DNA/polymer ratios above 1:2. As a representative example, FIG. 4shows the average effective diameters of particles formed with polymer 3as a function of polymer concentration. The diameters of the particlesvaried within the above range from experiment to experiment underotherwise identical conditions, possibly due to subtle differencesduring the stirring or addition of DNA solutions during complexformation (The order of addition of polymer and DNA solutions hadconsiderable impact on the nature of the resulting DNA/polymercomplexes. In order to form small particles, for example, it wasnecessary to add the DNA solution to a vortexing solution of polymer.For cases in which polymer solutions were added to DNA, only largemicron-sized aggregates were observed. Thus, it is possible that subtledifferences in stirring or rate of addition could be responsible forvariation in particle size). The ζ-potentials for complexes formed frompolymer 3 were on the order of +10 to +15 mV at DNA/polymer ratios above1:1, and the complexes did not aggregate extensively over an 18 hourperiod (pH=7, 25° C.) The positive ζ-potentials of these complexes maybe significant beyond the context of particle stability, as net positivecharges on particle surfaces may play a role in triggering endocytosis(Kabanov et al. Bioconjugate Chem. 6:7-20, 1995; Lim et al. J. Am. Chem.Soc. 122:6524-6525, 2000; Behr Chimia 51:34-36, 1997; Demeneix et al.,in Artificial Self-Assembling Systems for Gene Delivery (Feigner et al.,Eds.), American Chemical Society, Washington, D.C., 1996, pp. 146-151;Kabanov et al., in Self-Assembling Complexes for Gene Delivery: FromLaboratory to Clinical Trial, John Wiley and Sons, New York, 1998; eachof which is incorporated herein by reference).

[0203] Particles formed from polymer 3 were also relatively stable at37° C. For example, a sample of DNA/3 (DNA/3=1:5, average diameter=83nm) was incubated at 37° C. for 4 hours. After 4 hours, a bimodaldistribution was observed consisting of a fraction averaging 78 nm (>98%by number, 70% by volume) and a fraction of larger aggregates withaverage diameters of approximately 2.6 microns. These results suggestthat the degradation of complexes formed from polymer 3 occurred moreslowly than the degradation of polymer in solution, or that partialdegradation did not significantly affect the stability of the particles.The apparently increased stability of DNA/polymer complexes formed fromdegradable polycations relative to the degradation of the polymers insolution has also been observed for DNA/polymer complexes formed frompoly(4-hydroxy-L-proline ester) (Lim et al. J. Am. Chem. Soc.121:5633-5639, 1999; incorporated herein by reference).

[0204] The particle size and ζ-potential data in FIGS. 4 and 5 areconsistent with models of DNA condensation observed with otherpolycations (Kabanov et al. Bioconjugate Chem. 6:7-20, 1995; Putnam etal. Macromolecules 32:3658-3662, 1999; Lim et al. J. Am. Chem. Soc.121:5633-5639, 1999; Lim et al. J. Am. Chem. Soc. 122:6524-6525, 2000;Gonzalez et al. Bioconjugate Chem. 10:1068-1074, 1999; each of which isincorporated herein by reference). DNA is compacted into smallnegatively charged particles at very low polymer concentrations andparticle sizes increase with increasing polymer concentration (Accuratelight scattering data could not be obtained for DNA alone or forDNA/polymer associated species at DNA/polymer ratios lower than 1:0.5,since flexible, uncondensed DNA does not scatter light as extensively ascompacted DNA (Kabanov et al., in Self-Assembling Complexes for GeneDelivery. From Laboratory to Clinical Trial, John Wiley and Sons, NewYork, 1998; incorporated herein by reference).). Complexes reach amaximum diameter as charge neutrality is achieved and aggregationoccurs. Particle sizes decrease sharply at DNA/polymer concentrationsabove charge neutrality up to ratios at which additional polymer doesnot contribute to a reduction in particle diameter. This model isconfirmed by ζ-potential measurements made on complexes formed fromthese polymers. As shown in FIG. 5, the ζ-potentials of polymer/DNAparticles formed from polymer 3 were negative at low polymerconcentrations and charge neutrality was achieved near DNA/polymerratios of 1:0.75, resulting in extensive aggregation. The ζ-potentialsof the particles approached a limiting value ranging from +10 to +15 mVat DNA/polymer ratios above 1:2.

[0205] The average diameters of the complexes described above fallwithin the general size requirements for cellular endocytosis. We haveinitiated transfection experiments employing the NIH 3T3 cell line andthe luciferase reporter gene (pCMV-Luc). Thus far, polymers 1 and 2 haveshown no transfection activity in initial screening assays. By contrast,polymer 3 has demonstrated transfection efficiencies exceeding those ofPEI under certain conditions. Transfection experiments were performedaccording to the following general protocol: Cells were grown in 6-wellplates at an initial seeding density of 100,000 cells/well in 2 mL ofgrowth medium. Cells were grown for 24 hours after which the growthmedium was removed and replaced with 2 mL of serum-free medium.DNA/polymer complexes were formed as described in the ExperimentalSection (2 μg DNA, DNA/3=1:2 (w/w), 100 μL in MES (pH=6.0)] and added toeach well. DNA/PEI complexes were formed at a weight ratio of 1:0.75, aratio generally found in our laboratory to be optimal for PEItransfections. Transfections were carried out in serum-free medium for 4hours, after which medium was replaced with growth medium for 20additional hours. Relative transfection efficiencies were determinedusing luciferase (Promega) and cell protein assay (Pierce) kits. Resultsare expressed as relative light units (RLU) per mg of total cellprotein: for complexes of polymer 3, 1.07 (±0.43)×10⁶RLU/mg; for PEIcomplexes, 8.07 (±16)×10⁵ RLU/mg). No luciferase expression was detectedfor control experiments employing naked DNA or performed in the absenceof DNA. These transfection data are the results of initial screeningexperiments. These data suggest that polymers of this general structurehold promise as synthetic vectors for gene delivery and are interestingcandidates for further study. The relative efficacy of polymer 3relative to PEI is interesting, as our initial screening experimentswere performed in the absence of chloroquine and polymer 3 does notcurrently incorporate an obvious means of facilitating endosomal escape.It should be noted, however, that the pK_(a) values of the amines inthese polymers can be “tuned” to fall more directly within the range ofphysiologically relevant pH using this modular synthetic approach.Therefore, it will be possible to further engineer the “proton sponge”character (Behr Chimia 51:34-36, 1997; Demeneix et al., in ArtificialSelf-Assembling Systems for Gene Delivery (Felgner et al., Eds.),American Chemical Society, Washington, D.C., 1996, pp. 146-151; Kabanovet al., in Self-Assembling Complexes for Gene Delivery: From Laboratoryto Clinical Trial, John Wiley and Sons, New York, 1998; each of which isincorporated herein by reference) of these polymers, and thus enhancetheir transfection efficacies, directly through the incorporation of orcopolymerization with different diamine monomers.

SUMMARY

[0206] A general strategy for the preparation of new degradablepolymeric DNA transfection vectors is reported. Poly(β-amino esters) 1-3were synthesized via the conjugate addition ofN,N′-dimethylethylenediamine, piperazine, and4,4′-trimethylenedipiperidine to 1,4-butanediol diacrylate. The aminesin the bis(secondary amine) monomers actively participate inbond-forming processes during polymerization, obviating the need foramine protection/deprotection processes which characterize the synthesisof other poly(amino esters). Accordingly, this approach enables thegeneration of a variety of structurally diverse polyesters containingtertiary amines in their backbones in a single step from commerciallyavailable staring materials. Polymers 1-3 degraded hydrolytically inacidic and alkaline media to yield 1,4-butanediol and β-amino acids4a-6a and the degradation kinetics were investigated at pH 5.1 and 7.4.The polymers degraded more rapidly at pH 7.4 than at pH 5.1, consistentwith the pH/degradation profiles reported for other poly(amino esters).An initial screening assay designed to determine the effects of polymers1-3 on cell growth and metabolism suggested that these polymers andtheir hydrolytic degradation products were non-cytotoxic relative toPEI, a non-degradable cationic polymer conventionally employed as atransfection vector.

[0207] Polymers 1-3 interacted electrostatically with plasmid DNA atphysiological pH, initiating self-assembly processes that resulted innanometer-scale DNA/polymer complexes. Agarose gel electrophoresis,quasi-elastic dynamic light scattering (QELS), and zeta potentialmeasurements were used to determine the extent of the interactionsbetween the oppositely charged polyelectrolytes. All three polymers werefound to condense DNA into soluble DNA/polymer particles on the order of50-200 nm. Particles formed from polymers 1 and 2 aggregatedextensively, while particles formed from polymer 3 exhibited positiveζ-potentials (e.g., +10 to +15 mV) and did not aggregate for up to 18hours. The nanometer-sized dimensions and reduced cytotoxicities ofthese DNA/polymer complexes suggest that polymers 1-3 may be useful asdegradable polymeric gene transfection vectors. A thorough understandingof structure/activity relationships existing for this class of polymerwill expedite the design of safer polymer-based alternatives to viraltransfection vectors for gene therapy.

Example 2 Rapid, pH-Triggered Release from Biodegradable Poly(β-AminoEster) Microspheres Within the Ranger of Intracellular pH

[0208] Experimental Section

[0209] Fabrication of microspheres. The optimized procedure for thefabrication of microspheres was conducted in the following generalmanner: An aqueous solution of rhodamine-conjugated dextran (200 μL of a10 μg/μL solution, M_(n)≈70 kD) was suspended in a solution of poly-1 inCH₂Cl₂ (200 mg of poly-1 in 4 mL CH₂Cl₂, M_(n)≈10 kD), and the mixturewas sonicated for 10 seconds to form a primary emulsion. The cloudy pinkemulsion was added directly to a rapidly homogenized (5,000 rpm)solution of poly(vinyl alcohol) [50 mL, 1% PVA (w/w)] to form thesecondary emulsion. The secondary emulsion was homogenized for 30seconds before adding it to a second aqueous PVA solution [100 mL, 0.5%PVA (w/w)]. Direct analysis of the microsphere suspension using aCoulter microparticle analyzer revealed a mean particle size ofapproximately 5 micrometers. The secondary emulsion was stirred for 2.5hours at room temperature, transferred to a cold room (4° C.), andstirred for an additional 30 minutes. Microspheres were isolated at 4°C. via centrifugation, resuspended in cold water, and centrifuged againto remove excess PVA. The spheres were resuspended in water (15 mL) andlyophilized to yield a pink, fluffy powder. Characterization of thelyophilized microspheres was performed by optical, fluorescence, andscanning electron microscopies as described. Zeta potential wasdetermined using a Brookhaven Instruments ZetaPALS analyzer.

[0210] Discussion

[0211] Microparticles formed from biodegradable polymers are attractivefor use as delivery devices, and a variety of polymer-based microsphereshave been employed for the sustained release of therapeutic compounds(Anderson Nature 392(Suppl.):25-30, 1996; Friedman Nature Med.2:144-147, 1996; Crystal Science 270:404-410, 1995; Mulligan Science260:926-932, 1993; Luo et al. Nat. Biotechnol. 18:33-37,2000; Behr Acc.Chem. Res. 26:274-278, 1993; each of which is incorporated herein byreference). However, for small-molecule-, protein-, and DNA-basedtherapeutics that require intracellular administration and traffickingto the cytoplasm, there is an increasing demand for new materials thatfacilitate triggered release in response to environmental stimuli suchas pH (Zauner et al. Adv. Drug Del. Rev. 30:97-113, 1998; incorporatedherein by reference). Following endocytosis, the pH within cellularendosomal compartments is lowered, and foreign material is degraded uponfusion with lysosomal vesicles (Kabanov et al. Bioconjugate Chem.6:7-20, 1995; incorporated herein by reference). New materials thatrelease molecular payloads upon changes in pH within the intracellularrange and facilitate escape from hostile intracellular environmentscould have a fundamental and broad-reaching impact on the administrationof hydrolytically- and/or enzymatically-labile drugs (Zauner et al. Adv.Drug Del. Rev. 30:97-113, 1998; Kabanov et al. Bioconjugate Chem.6:7-20, 1995; each of which is incorporated herein by reference).Herein, the fabrication of pH-responsive polymer micro spheres thatrelease encapsulated contents quantitatively and essentiallyinstantaneously upon changes in pH within the intracellular range isreported.

[0212] The synthesis of poly(β-amino ester) 1 has been described abovein Example 1 (Miller Angew. Chem. Int. Ed. 37:1768-1785, 1998; Hope etal. Molecular Membrane Technology 15: 120 14, 1998; Deshmukh et al. NewJ. Chem. 21:113-124, 1997; each of which is incorporated herein byreference). Poly-1 is hydrolytically degradable, was non-cytotoxic ininitial screening assays, and is currently under investigation as asynthetic vector for DNA delivery in gene therapy applications. Thesolubility of the polymer in aqueous media is directly influenced bysolution pH. Specifically, the solid, unprotonated polymer is insolublein aqueous media in the pH range 7.0 to 7.4, and the transition betweensolubility and insolubility occurs at a pH around 6.5. Based on thedifferences between extracellular and endosomal pH (7.4 and 5.0-6.5,respectively), we hypothesized that microspheres formed from poly-1might be useful for the encapsulation and triggered release of compoundswithin the range of intracellular pH.

[0213] The encapsulation of therapeutic compounds within polymermicrospheres is often achieved employing a double emulsion process(O'Donnell et al. Adv. Drug Delivery Rev. 28:25-42, 1997; incorporatedherein by reference). The double emulsion process is well establishedfor the fabrication of microspheres from hydrophobic polymers such aspoly(lactic-co-glycolic acid) (PLGA), a biodegradable polymerconventionally employed in the development of drug delivery devices(Anderson et al. Adv. Drug Delivery Rev. 28:5-24, 1997; Okada Adv. DrugDelivery Rev. 28:43-70, 1997; each of which is incorporated herein byreference). Preliminary experiments demonstrated the feasibility of thedouble emulsion process for the encapsulation of water-soluble compoundsusing poly-1. Rhodamine-conjugated dextran was chosen as a model forsubsequent encapsulation and release studies for several reasons: 1)rhodamine is fluorescent, allowing loading and release profiles to bedetermined by fluorescence spectroscopy, 2) loaded microspheres could beimaged directly by fluorescence microscopy, and 3) the fluorescenceintensity of rhodamine is relatively unaffected by pH within thephysiological range (Haugland, Handbook of Fluorescent Probes andResearch Chemicals, 6th ed., Molecular Probes, Inc., 1996, p. 29;incorporated herein by reference).

[0214] Microspheres encapsulating labeled dextran were fabricated frompoly-1 and compared to controls formed from PLGA. The size distributionsof microspheres formed from poly-1 correlated well with thedistributions of PLGA microspheres within the range of 5-30 μm. Averageparticle sizes could be controlled by variations in experimentalparameters such as homogenization rates and aqueous/organic solventratios (O'Donnell et al. Adv. Drug Delivery Rev. 28:25-42, 1997;incorporated herein by reference). In contrast to PLGA microspheres,however, spheres formed from poly-1 aggregated extensively duringcentrifugation and washing steps (see Experimental Section above).Microspheres resuspended at pH 7.4 consisted primarily of largeaggregates, and scanning electron microscopy (SEM) images revealedclusters of spheres that appeared to be physically joined or “welded”(data not shown).

[0215] It was found that aggregation could be eliminated ifcentrifugation and washing were conducted at reduced temperatures (4°C.), presumably due to the hardening of the polymer spheres at thislower temperature. SEM images of dextran-loaded poly-1 microspheresprepared in the 8-10 μm range revealed significant fracturing and theformation of large holes on their surfaces. Microspheres targeted in therange of 4-6 μm, however, were essentially free of cracks, holes, andother defects (FIG. 6). Microspheres formulated for subsequent releaseexperiments were fabricated in the smaller (<6 μm) range. Encapsulationefficiencies for loaded poly-1 microspheres, determined by dissolvingthe spheres at pH 5.1 and measuring fluorescence intensity, were as highas 53%.

[0216] Suspensions of dried poly-1 microspheres at pH=7.4 consistedprimarily of single, isolated microspheres as determined by optical andfluorescence microscopy (FIG. 8a). The zeta potential (ζ) ofmicroparticle suspensions of poly-1 microspheres at pH 7 was +3.75(+0.62) mV, suggesting that the surfaces of the microspheres carry anoverall positive charge at physiological pH. This could be relevant tothe targeting of these microspheres for cellular uptake, because netpositive charges on particle surfaces may play a role in triggeringendocytosis (Zauner et al. Adv. Drug Delivery Rev. 30:97-113, 1998;incorporated herein by reference).

[0217] Poly-1 microspheres suspended at pH 7.4 remained stable towardaggregation and degradation for several weeks (by visual inspection),but the microspheres dissolved instantly when the pH of the suspendingmedium was lowered between 5.1 and 6.5.

[0218] The release of labeled dextran from poly-1 microspheres wasdetermined quantitatively by fluorescence microscopy (FIG. 7). Therelease profile at pH 7.4 was characterized by a small initial burst influorescence (7-8%) which reached a limiting value of about 15% after 48hours. This experiment demonstrated that the degradation of poly-1 wasrelatively slow under these conditions and that greater than 90% ofencapsulated material could be retained in the polymer matrix forsuitably long periods of time at physiological pH.

[0219] To examine the application of poly-1 microspheres to thetriggered release of encapsulated drugs in the endosomal pH range, weconducted a similar experiment in which the pH of the suspension mediumwas changed from 7.4 to 5.1 during the course of the experiment. Asshown in FIG. 7, the microspheres dissolved rapidly when the suspensionbuffer was exchanged with acetate buffer (0.1 M, pH=5.1), resulting inessentially instantaneous and quantitative release of the labeleddextran remaining in the polymer matrices. In sharp contrast, therelease from dextran-loaded PLGA microspheres did not increase for up to24 hours after the pH of the suspending medium was lowered (FIG. 7).FIG. 8 shows fluorescence microscopy images of: (a) a sample ofdextran-loaded microspheres at pH 7.4; and (b) a sample to which a dropof acetate buffer was added at the upper right edge of the microscopecoverslip. The rapid release of rhodamine-conjugated dextran wasvisualized as streaking extending from the dissolving microspheres inthe direction of the diffusion of added acid and an overall increase inbackground fluorescence (elapsed time≈5 seconds).

[0220] When targeting therapeutic compounds for intracellular deliveryvia endocytosis or phagocytosis, it is not only important to consider ameans by which the drug can be released from its carrier, but also ameans by which the drug can escape endosomal compartments prior to beingrouted to lysosomal vesicles (Luo et al. Nat. Biotechnol. 18:33-37,2000; Zauner et al. Adv. Drug Delivery Rev. 30:97-113, 1998; each ofwhich is incorporated herein by reference). One strategy forfacilitating endosomal escape is the incorporation of weak bases, or“proton sponges,” which are believed to buffer the acidic environmentwithin an endosome and disrupt endosomal membranes by increasing theinternal osmotic pressure within the vesicle (Demeneix et al., inArtificial Self-Assembling Systems for Gene Delivery (Felgner et al.,Eds.), American Chemical Society, Washington, D.C., 1996, pp. 146-151;incorporated herein by reference). Poly-1 microspheres are capable ofreleasing encapsulated material in the endosomal pH range via amechanism (dissolution) that involves the protonation of amines in thepolymer matrix. Thus, in addition to the rapid release of drug, poly-1microspheres may also provide a membrane-disrupting means of endosomalescape, enhancing efficacy by prolonging the lifetimes of hydrolyticallyunstable drugs contained in the polymer matrix.

[0221] Microspheres fabricated from poly-1 could represent an importantaddition to the arsenal of pH-responsive materials applied forintracellular drug delivery, such as pH-responsive polymer/liposomeformulations (Gerasimov et al. Adv. Drug Delivery Rev. 38:317-338, 1999;Linhart et al. Langmuir 16:122-127, 2000; Linhardt et al. Macromolecules32:4457-4459, 1999; each of which is incorporated herein by reference).In contrast to many liposomal formulations, polymer microspheres arephysically robust and can be stored dried for extended periods withoutdeformation, decomposition, or degradation (Okada Adv. Drug DeliveryRev. 28:43-70, 1997; incorporated herein by reference)—an importantconsideration for the formulation and packaging of new therapeuticdelivery systems. The microspheres investigated in this current studyfall within the size range of particles commonly used to target deliveryto macrophages (Hanes et al. Adv. Drug Delivery Rev. 28:97-119, 1997;incorporated herein by reference). The rapid pH-release profiles for thepoly-1 microspheres described above may therefore be useful in thedesign of new DNA-based vaccines which currently employ PLGA as anencapsulating material (Singh et al. Proc. Natl. Acad. Sci. USA97:811-816, 2000; Andoet al. J. Pharm. Sci. 88:126-130, 1999; Hedley etal. Nat. Med. 4:365-368, 1998; each of which is incorporated herein byreference).

Example 3 Accelerated Discovery of Synthetic Transfection Vectors:Parallel Synthesis and Screening of a Degradable Polymer Library

[0222] Introduction

[0223] The safe and efficient delivery of therapeutic DNA to cellsrepresents a fundamental obstacle to the clinical success of genetherapy (Luo et al. Nat. Biotechnol. 18:33-37, 2000; Anderson Nature 392Suppl.:25-30, 1996; each of which is incorporated herein by reference).The challenges facing synthetic delivery vectors are particularly clear,as both cationic polymers and liposomes are less effective at mediatinggene transfer than viral vectors. The incorporation of new designcriteria has led to recent advances toward functional delivery systems(Lim et al. J. Am. Chem. Soc. 123:2460-2461, 2001; Lim et al. J. Am.Chem. Soc. 122:6524-6525, 2000; Hwang et al. Bioconjugate Chem.12:280-290, 2001; Putnam et al. Proc. Natl. Acad. Sci. USA 98:1200-1205,2001; Benns et al. Bioconjugate Chem. 11:637-645, 2000; Midoux et al.Bioconjugate Chem. 10:406-411, 1999; each of which is incorporatedherein by reference). However, the paradigm for the development ofpolymeric gene delivery vectors remains the incorporation of thesedesign elements into materials as part of an iterative, linearprocess—an effective, albeit slow, approach to the discovery of newvectors. Herein, we report a parallel approach suitable for thesynthesis of large libraries of degradable cationic polymers andoligomers and the discovery of new synthetic vector families throughrapid cell-based screening assays (for a report on the parallelsynthesis and screening of degradable polymers for tissue engineering,see: Brocchini et al. J. Am. Chem. Soc. 119:4553-4554, 1997;incorporated herein by reference).

[0224] Experimental Section

[0225] General Considerations. All manipulations involving live cells orsterile materials were performed in a laminar flow hood using standardsterile technique. Gel permeation chromatography (GPC) was performedusing a Hewlett Packard 1100 Series isocratic pump, a Rheodyne Model7125 injector with a 100-μL injection loop, and two PL-Gel mixed-Dcolumns in series (5 μm, 300×7.5 mm, Polymer Laboratories, Amherst,Mass.). THF/0.1 M piperidine was used as the eluent at a flow rate of1.0 mL/min. Data was collected using an Optilab DSP interferometricrefractometer (Wyatt Technology, Santa Barbara, Calif.) and processedusing the TriSEC GPC software package (Viscotek Corporation, Houston,Tex.). The molecular weights and polydispersities of the polymers arereported relative to monodisperse polystyrene standards.

[0226] Materials. Primary amine and secondary amine monomers 1-20 werepurchased from Aldrich Chemical Company (Milwaukee, Wis.), Lancaster(Lancashire, UK), Alfa Aesar Organics (Ward Hill, Mass.), and Fluka(Milwaukee, Wis.). Diacrylate monomers A-G were purchased fromPolysciences, Inc. (Warrington, Pa.), Alfa Aesar, and Scientific PolymerProducts, Inc. (Ontario, N.Y.). All monomers were purchased in thehighest purity available (from 97% to 99+%) and were used as receivedwithout additional purification. Plasmid DNA containing the fireflyluciferase reporter gene (pCMV-Luc) was purchased from ElimBiopharmaceuticals, Inc. (San Francisco, Calif.) and used withoutfurther purification.(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) waspurchased from Sigma Chemical Company (St. Louis, Mo.). Monkey kidneyfibroblasts (COS-7 cells) used in transfection assays were purchasedfrom American Type Culture Collection (Manassas, Va.) and grown at 37°C., 5% CO₂ in Dulbecco's modified Eagle's medium, 90%; fetal bovineserum, 10%; penicillin, 100 units/mL; streptomycin, 100 μg/mL.Luciferase detection kits used in high-throughput transfection assayswere purchased from Tropix (Bedford, Mass.). All other materials andsolvents were used as received without additional purification.

[0227] Synthesis of Polymer Library. All 140 polymerization reactionswere conducted simultaneously as an array of individually labeled vialsaccording to the following general protocol. Individual exceptions arenoted where appropriate. Amine monomers 1-20 (2.52 mmol) were chargedinto appropriately labeled vials (as shown below): liquid monomers weremeasured and transferred quantitatively using microliter pipettes; solidmonomers were weighed directly into each vial. Anhydrous CH₂Cl₂ (1 mL)was added to each vial. One equivalent of liquid diacrylates A-F (2.52mmol) was added to each appropriate reaction vial using a microliterpipette, and the vial was capped tightly with a Teflon-lined cap. Oneequivalent of semi-solid diacrylate G was added to the appropriate vialsas a solution in CH₂Cl₂ (2.52 mmol, 1 mL of a 2.52M solution in CH₂Cl₂)and the vials were tightly capped. An additional aliquot of CH₂Cl₂ (2mL) was added to the reaction vials containing 19 and 20 to aid in thesolubility of these monomers. The tightly capped vials were arrayed intwo plastic test tube racks and secured to an orbital shaker in a 45° C.oven. (CAUTION: The heating of capped vials represents a possibleexplosion hazard. Oven temperature was monitored periodically for oneweek prior to the experiment to ensure reliable thermal stability.Temperatures were found to vary within +/−1° C. during this time period.Several test vials were monitored prior to conducting the largerexperiment). The reaction vials were shaken vigorously at 45° C. for 5days and allowed to cool to room temperature. Vials were placed in alarge dessicator and placed under aspirator vacuum for 1 day and highvacuum for an additional 5 days to ensure complete removal of solvent.The samples obtained were analyzed by GPC (55% of total library, seeTable 2) and used directly in all subsequent screening experiments.TABLE 2 GPC survey of 55% of the screening library showing molecularweights (M_(w)) and polydispersities (shown in parentheses). A B C D E FG  1 5900 4725 5220 1690 (1.93) (1.89) (1.95) (1.74)  2 6920 6050 5640(1.87) (1.78) (1.85)  3 6690 6050 2060 (1.79) (1.78) (1.76)  4 7810 57209720 7960 7940 (2.49) (2.20) (2.49) (4.08) (3.25)  5 10 800 5000 15 30017 200 15 300 Insol. 9170 (2.75) (2.50) (3.17) (6.91) (3.92) (2.50)  621 000 10 200 18 000 (3.70) (3.4) (6.06)  7 14 300 11 880 20 200 10 30015 500 22 500 (3.25) (3.3) (3.44) (4.26) (4.89) (3.92)  8 2310 11 5202230 (1.62) (3.60) (1.73)  9 1010 2505 1240 Insol. (1.33) (1.67) (1.16)10 <1000 Insol. 11 6800 Insol. 9440 5550 6830 1990 6420 (1.91) (1.79)(2.23) (1.93) (1.43) (1.75) 12 9310 9100 11 900 5810 12 300 (2.06)(2.53) (2.18) (1.77) (1.85) 13 2990 3180 3680 2550 3230 3580 (1.64)(2.12) (1.64) (1.82) (1.82) (1.64) 14 1350 3180 2110 1400 1752 2025(1.35) (2.12) (1.69) (1.4) (1.46) (1.62) 15 1550 (1.51) 16 16 380 (2.60)17 8520 7290 (2.13) (1.94) 18 <1000 19 12 400 18 445 39 700 17 400 14800 13 900 (2.28) (2.17) (1.90) (1.93) (1.98) (1.86) 20 16 900 46 060 49600 30 700 18 700 17 100 (2.40) (3.29) (2.25) (2.72) (2.72) (2.22)

[0228] Determination of Water Solubility. The solubilities of allsamples sample were determined simultaneously at a concentration of 2mg/mL in the following general manner. Each polymer sample (5 mg) wasweighed into a 12 mL scintillation vial and 2.5 mL of acetate buffer (25mM, pH=5.0) was added to each sample using an a pipettor. Samples wereshaken vigorously at room temperature for 1 hour. Each sample wasobserved visually to determine solubility.

[0229] Agarose Gel Electrophoresis Assay. The agarose gelelectrophoresis assay used to determine the ability of polymers to formcomplexes with DNA was performed in the following manner. Using thesolutions prepared in the above solubility assay (2 mg/mL in acetatebuffer, 25 mM, pH=5.0), stock solutions of the 70 water-soluble polymerswere arrayed into a 96-well cell culture plate. DNA/polymer complexeswere formed at a ratio of 1:5 (w/w) by transferring 10 μL of eachpolymer solution from the stock plate to a new plate using amultichannel pipettor. Each polymer was further diluted with 90 μL ofacetate buffer (25 mM, pH=5.0, total volume=100 μL) and the plate wasshaken for 30 seconds on a mechanical shaker. An aqueous solution ofplasmid DNA (100 μL of a 0.04 μg/μL solution) was added to each well inthe plate and the solutions were vigorously mixed using a multichannelpipettor and a mechanical shaker. DNA/polymer complexes were formed at aratio of 1:20 (w/w) in the same manner with the following exceptions: 40μL of polymer stock solution was transferred to a new plate and dilutedwith 60 μL of acetate buffer (total volume=100 μL) prior to adding theaqueous DNA solution (100 μL). DNA/polymer complexes were incubated atroom temperature for 30 minutes, after which samples of each solution(15 μL) were loaded into a 1% agarose gel (HEPES, 20 mM, pH=7.2, 500ng/mL ethidium bromide) using a multichannel pipettor. NOTE: Sampleswere loaded on the gel with a loading buffer consisting of 10% Ficoll400 (Amersham Pharmacia Biotech, Uppsala, Sweden) in HEPES (25 mM,pH=7.2). Bromphenol blue was not included as a visual indicator in theloading buffer, since this charged dye appeared to interfere with thecomplexation of polymer and DNA. Samples were loaded according to thepattern shown in FIG. 9, such that samples corresponding to DNA/polymerratios of 1:5 and 1:20 were assayed in adjacent positions on the gel.The gel was run at 90V for 30 minutes and DNA bands were visualized byethidium bromide staining.

[0230] General Protocol for Cell Transfection Assays. Transfectionassays were performed in triplicate in the following general manner.COS-7 cells were grown in 96-well plates at an initial seeding densityof 15,000 cells/well in 200 μL of phenol red-free growth medium (90%Dulbecco's modified Eagle's medium, 10% fetal bovine serum, penicillin100 units/mL, streptomycin 100 μg/mL). Cells were grown for 24 hours inan incubator, after which the growth medium was removed and replacedwith 200 μL of Optimem medium (Invitrogen Corp., Carlsbad, Calif.)supplemented with HEPES (total concentration=25 mM). Polymer/DNAcomplexes prepared from the 56 water-soluble/DNA-complexing polymerspreviously identified were prepared as described above at a ratio of1:20 (w/w)) using a commercially available plasmid containing thefirefly luciferase reporter gene (pCMV-Luc). An appropriate volume ofeach sample was added to the cells using a multichannel pipettor (e.g.,15 μL yielded 300 ng DNA/well; 30 μL yielded 600 ng DNA/well). Controlsemploying poly(ethylene imine) (PEI) and polylysine, prepared atDNA/polymer ratios of 1:1, were prepared in a similar manner andincluded with DNA and no-DNA controls. Controls employing Lipofectamine2000 (Invitrogen Corp.) were performed at several concentrations (0.1,0.2, 0.4, and 0.6 pL) as described in the technical manual for thisproduct (http://lifetechnologies.com). Cells were incubated for 4 hours,after which the serum-free growth medium was removed and replaced with100 μL of phenol-red-free growth medium. Cells were incubated for anadditional period of time (typically varied between 36 to 60 hours) andluciferase expression was determined using a commercially availableassay kit (Tropix, Inc., Bedford, Mass.). Luminescence was quantified inwhite, solid-bottom polypropylene 96-well plates using a 96-wellbioluminescence plate reader. Luminescence was expressed in relativelight units and was not normalized to total cell protein in this assay.

[0231] Results and Discussion

[0232] Poly(β-amino ester)s are hydrolytically degradable, condenseplasmid DNA at physiological pH, and are readily synthesized via theconjugate addition of primary or secondary amines to diacrylates (Eq. 1and 2) (Lynn et al. J. Am. Chem. Soc. 122:10761-10768, 2000;incorporated herein by reference). An initial screen of model polymersidentified these materials as potential gene carriers and demonstratedthat structural variations could have a significant impact on DNAbinding and transfection efficacies (Lynn et al. J. Am. Chem. Soc.122:10761-10768, 2000; incorporated herein by reference). We reasonedthat this approach provided an attractive framework for the elaborationof large libraries of structurally-unique polymers for severalreasons: 1) diamine and diacrylate monomers are inexpensive,commercially available starting materials, 2) polymerization can beaccomplished directly in a single synthetic step, and 3) purificationsteps are generally unnecessary as no byproducts are generated duringpolymerization.

[0233] The paucity of commercially available bis(secondary amines)limits the degree of structural diversity that can be achieved using theabove synthetic approach. However, the pool of useful, commerciallyavailable monomers is significantly expanded when primary amines areconsidered as potential library building blocks. Because the conjugateaddition of amines to acrylate groups is generally tolerant offunctionalities such as alcohols, ethers, and tertiary amines (Ferrutiet al. Adv. Polym. Sci. 58:55-92, 1984; incorporated herein byreference), we believed that the incorporation of functionalized primaryamine monomers into our synthetic strategy would serve to broadenstructural diversity. Diacrylate monomers A-G and amine monomers 1-20were selected for the synthesis of an initial screening library.

[0234] The size of the library constructed from this set of monomers (7diacrylates×20 amines 140 structurally-unique polymers) was chosen to belarge enough to incorporate sufficient diversity, yet small enough to bepractical without the need for automation in our initial studies. It wasunclear at the outset whether a polymer such as G16 (formed fromhydrophobic and sterically bulky monomers G and 16) would bewater-soluble at physiological pH or be able to condense DNAsufficiently. However, monomers of this type were deliberatelyincorporated to fully explore diversity space, and in anticipation thatthis library may ultimately be useful as a screening population for thediscovery of materials for applications other than gene delivery (For areport on the parallel synthesis and screening of degradable polymersfor tissue engineering, see: Brocchini et al. J. Am. Chem. Soc.119:4553-4554, 1997, incorporated herein by reference; Lynn et al.Angew. Chem. Int. Ed. 40:1707-1710, 2001; incorporated herein byreference).

[0235] Polymerization reactions were conducted simultaneously as anarray of individually labeled vials. Reactions were performed inmethylene chloride at 45° C. for 5 days, and polymers were isolated byremoval of solvent to yield 600-800 mg of each material. Reactionsperformed on this scale provided amounts of each material sufficient forroutine analysis by GPC and all subsequent DNA-binding, toxicity, andtransfection assays. A survey of 55% of the library by GPC indicatedmolecular weights ranging from 2000 to 50 000 (relative to polystyrenestandards). As high molecular weights are not required forDNA-complexation and transfection (as shown below) (Kabanov et al., inSelf-Assembling Complexes for Gene Delivery: From Laboratory to ClinicalTrial, John Wiley and Sons, New York, 1998; incorporated herein byreference), this library provided a collection of polymers and oligomerssuitable for subsequent screening assays.

[0236] Of the 140 members of the screening library, 70 samples weresufficiently water-soluble (2 mg/mL, 25 mM acetate buffer, pH=5.0) to beincluded in an electrophoretic DNA-binding assay (FIG. 9). To performthis assay as rapidly and efficiently as possible, samples were mixedwith plasmid DNA at ratios of 1:5 and 1:20 (DNA/polymer, w/w) in 96-wellplates and loaded into an agarose gel slab capable of assaying up to 500samples using a multi-channel pipettor. All 70 water-soluble polymersamples were assayed simultaneously at two different DNA/polymer ratiosin less than 30 minutes. As shown in FIG. 9, 56 of the 70 water-solublepolymer samples interacted sufficiently with DNA to retard migrationthrough the gel matrix (e.g., A4 or A5), employing the 1:20 DNA/polymerratio as an exclusionary criterion. Fourteen polymers were discardedfrom further consideration (e.g., A7 and A8), as these polymers did notcomplex DNA sufficiently.

[0237] The DNA-complexing materials identified in the above assay werefurther investigated in transfection assays employing plasmid DNA andthe COS-7 cell line. All assays were performed simultaneously with thefirefly luciferase reporter gene (pCMV-Luc) and levels of expressedprotein were determined using a commercially available assay kit and a96-well luminescence plate reader. FIG. 10 displays data generated froman assay employing pCMV-Luc (600 ng/well) at DNA/poly ratios of 1:20(w/w). The majority of the polymers screened did not mediatetransfection above a level typical of “naked” DNA (no polymer) controlsunder these conditions. However, several wells expressed higher levelsof protein and the corresponding polymers were identified as “hits” inthis assay. Polymers B14 (M_(w)=3180) and G5 (M_(w)=9170), for example,yielded transfection levels 4-8 times higher than control experimentsemploying poly(ethylene imine) (PEI), a polymer conventionally employedas a synthetic transfection vector (Boussif et al. Proc. Natl. Acad.Sci. USA 92:7297-7301, 1995; incorporated herein by reference), andtransfection levels within or exceeding the range of expressed proteinusing Lipofectamine 2000 (available from Invitrogen Corp. (Carlsbad,Calif.)), a leading commercially available lipid-based transfectionvector system. Polymers A14, C5, G7, G10, and G12 were also identifiedas positive “hits” in the above experiment, but levels of geneexpression were lower than B14 and G5.

[0238] Structural differences among synthetic polymers typicallypreclude a general set of optimal transfection conditions. For example,polymers C5, C14, and G14 were toxic at the higher concentrationsemployed above (Determined by the absence of cells in wells containingthese polymers as observed upon visual inspection. These polymers wereless toxic and mediated transfection at lower concentration.), butmediated transfection at lower DNA and polymer concentrations (data notshown). The assay system described above can easily be modified toevaluate polymers as a function of DNA concentration, DNA/polymer ratio,cell seeding densities, or incubation times. Additional investigationwill be required to more fully evaluate the potential of this screeninglibrary, and experiments to this end are currently underway.

[0239] The assays above were performed in the absence of chloroquine, aweak base commonly added to enhance in vitro transfection (Putnam et al.Proc. Natl. Acad. Sci. USA 98:1200-1205, 2001; Benns et al. BioconjugateChem. 11:637-645, 2000; Midoux et al. Bioconjugate Chem. 10:406-411,1999; Kabanov et al., in Self-Assembling Complexes for Gene Delivery:From Laboratory to Clinical Trial, John Wiley and Sons, New York, 1998;each of which is incorporated herein by reference), and the resultstherefore reflect the intrinsic abilities of those materials to mediatetransfection. The polymers containing monomer 14 are structurallysimilar to other histidine containing “proton sponge” polymers (Putnamet al. Proc. Natl. Acad. Sci. USA 98:1200-1205, 2001; Benns et al.Bioconjugate Chem. 11:637-645, 2000; Midoux et al. Bioconjugate Chem.10:406-411, 1999; each of which is incorporated herein by reference),and could enhance transfection by buffering acidic intracellularcompartments and mediating endosomal escape (Putnam et al. Proc. Natl.Acad. Sci. USA 98:1200-1205, 2001; Benns et al. Bioconjugate Chem.11:637-645, 2000; Midoux et al. Bioconjugate Chem. 10:406-411, 1999;Boussif et al. Proc. Natl. Acad. Sci. USA 92:7297-7301, 1995; each ofwhich is incorporated herein by reference). The efficacy of polymerscontaining monomer 5 is surprising in this context, as these materialsdo not incorporate an obvious means of facilitating endosomal escape.While the efficacy of these latter polymers is not yet understood, theirdiscovery helps validate our parallel approach and highlights the valueof incorporating structural diversity, as these polymers may not havebeen discovered on an ad hoc basis. Polymers incorporating hydrophilicdiacrylates D and E have not produced “hits” under any conditions thusfar, providing a possible basis for the development of more focusedlibraries useful for the elucidation of structure/activityrelationships.

[0240] We have generated a library of 140 degradable polymers andoligomers useful for the discovery of new DNA-complexing materials andgene delivery vectors. Several of these materials are capable ofcondensing DNA into structures small enough to be internalized by cellsand release the DNA in a transcriptionally active form. The total timecurrently required for library design, synthesis, and initial screeningassays is approximately two weeks. However, the incorporation ofrobotics and additional monomers could significantly accelerate the paceat which new DNA-complexing materials and competent transfection vectorsare identified.

Example 4 Semi-Automated Synthesis and Screening of a Large Library ofDegradable Cationic Polymers for Gene Delivery

[0241] One of the major barriers to the success of gene therapy in theclinic is the lack of safe and efficient methods of delivering nucleicacids. Currently, the majority of clinical trials use modified virusesas delivery vehicles, which, while effective at transferring DNA tocells, suffer from potentially serious toxicity and production problems(Somia et al. Nat Rev Genet. 1:91 (2000); incorporated herein byreference). In contrast, non-viral systems offer a number of potentialadvantages, including ease of production, stability, low immunogenicityand toxicity, and reduced vector size limitations (Ledley Human GeneTherapy 6:1129 (1995); incorporated herein by reference). Despite theseadvantages, however, existing non-viral delivery systems are far lessefficient than viral vectors (Luo et al. Nature Biotechnology 18:33(2000); incorporated herein by reference).

[0242] One promising group of non-viral delivery compounds are cationicpolymers, which spontaneously bind and condense DNA. A wide variety ofcationic polymers that transfect in vitro have been characterized, bothnatural, such as protein (Fominaya et al. Journal of BiologicalChemistry 271:10560 (1996); incorporated herein by reference) andpeptide systems (Schwartz et al. Curr Opin Mol Ther. 2:162 (2000);incorporated herein by reference), and synthetic polymers such aspoly(ethylene imine) (Boussif et al. Proceedings of the National Academyof Sciences of the United States of America 92:7297 (1995);incorporatedherein by reference) and dendrimers (Kabanov et al. Self-assemblingcomplexes for gene delivery: from laboratory to clinical trial, Wiley,Chichester; N.Y., 1998; incorporated herein by reference). Recentadvances in polymeric gene delivery have in part focused on making thepolymers more biodegradable to decrease toxicity. Typically, thesepolymers contain both chargeable amino groups, to allow for ionicinteractions with the negatively charged phosphate backbone of nucleicacids, and a biodegradable linkage such as a hydrolyzable ester linkage.Several examples of these include poly(alpha-(4-aminobutyl)-L-glycolicacid) (Lim et al. Journal of the American Chemical Society 122:6524(2000); incorporated herein by reference), network poly(amino ester)(Lim et al. Bioconjugate Chemistry 13:952 (2002); incorporated herein byreference), and poly(beta-amino ester)s (Lynn et al. Journal of theAmerican Chemical Society 122:10761 (2000); Lynn et al. Journal of theAmerican Chemical Society 123:8155 (2001); each of which is incorporatedherein by reference). Poly(beta-amino ester)s are particularlyinteresting because they show low cytotoxicity and are easilysynthesized via the conjugate addtion of a primary amine orbis(secondary amine) to a diacrylate (FIG. 11) (Lynn et al. Journal ofthe American Chemical Society 122:10761 (2000); Lynn et al. Journal ofthe American Chemical Society 123:8155 (2001); each of which isincorporated herein by reference).

[0243] Traditional development of new biomedical polymers has been aniterative process. Polymers were typically designed one at a time andthen individually tested for their properties. More recently, attentionhas focused on the development of parallel, combinatorial approachesthat facilitate the generation of structurally-diverse libraries ofpolymeric biomaterials (Brocchini Advanced Drug Delivery Reviews 53:123(2001); incorporated herein by reference). This combinatorial approachhas also been applied to the discovery of gene delivery polymers. Forexample, Murphy et al. generated a targeted combinatorial library of 67peptoids via solid-phase synthesis and screened them to identify newgene delivery agents (Murphy et al. Proceedings of the National Academyof Sciences of the United States of America 95:1517 (1998); incorporatedherein by reference).

[0244] In this Example is described new tools for high-throughput,parallel combinatorial synthesis and cell-based screening of a largelibrary of 2350 structurally diverse, poly(beta-amino ester)s. Thisapproach allows for the screening of polymers in cell-based assayswithout the polymers ever leaving the solution phase followingsynthesis. This approach combined with the use of robotic fluid handlingsystems allows for the generation and testing of thousands of syntheticpolymers in cell-based assays in a relatively short amount of time.Using this approach, 46 new polymers that perform as well or better thanconventional non-viral delivery systems such as poly(ethylene imine)have been identified.

[0245] Results and Discussion

[0246] High-throughput polymer synthesis. The primary factors limitingthe throughput and automation of poly(beta-amino esters) synthesis andtesting was the viscosity of monomer and polymer solutions, and thedifficulty with manipulating the solid polymer products. Whileautomation of liquid handling is straightforward using conventionalrobotics, the manipulation of solids and viscous liquids on a smallscale is not. Therefore, a system was developed in which polymers couldbe synthesized and screened in cell-based assays without leaving thesolution phase. Since this would require the presence of residualsolvent in the cell assays, the relatively non-toxic solvent, dimethylsulfoxide (DMSO) was chosen. DMSO is a commonly used solvent in cellculture and is routinely used in storing frozen stocks of cells. DMSO ismiscible with water and is generally well tolerated in living systems.

[0247] The first step in preparing for high-throughput synthesis was toidentify conditions that would allow for the production of polymer yetpossess a manageable viscosity. Small scale, pilot experiments showedthat polymerization could be performed effectively at 1.6 M in DMSO at56° C. for 5 days. Based on these experiments, a general strategy wasdeveloped for polymer synthesis and testing. All monomers (FIG. 12) werediluted to 1.6 M in DMSO, and then using both a fluid-handling robot anda 12 channel micropipettor, we added 150 microliters of each amine anddiacrylate monomer into a polypropylene deep well plate and then sealedit with aluminum foil. The plates were placed on an orbital shaker andincubated at 56° C. for 5 days. To compensate for the increasedviscosity of polymeric solutions, 1 ml of DMSO was added to each well ofthe plate, and the plates were then stored at 4° C. until further use.These methods allos for 2350 reactions in a single day. Furthermore, theproduction and storage of polymers in a 96-well format allowed for aneasy transition into 96-well format cell-based testing of polymertransfection efficiency.

[0248] High-throughput polymer testing. Once synthesized, all polymerswere tested for their ability to deliver the Lucieferase expressingplasmid, pCMV-luc, into the monkey kidney fibroblast cell line COS-7.Due to the large size of the polymer library, a high-throughput methodfor cell based screening of transfection efficience was developed. Sincepolymers were stored in 96 well plates, all polymer dilutions, DNAcomplexation, and cell transfections were performed in parallel bydirectly transferring polymers from plate to plate using a liquidhandling robot. All polymers were synthesized using the sameconcentration of amine monomer, thus comparison between polymers at afixed amine monomer:DNA phosphate ratio (N:P ratio) was straightforward.While the amine monomers contain either one, two, or three amines permonomer, initial broad-based screens for transfection efficiency weregreatly simplified by maintaining a constant monomer concentration inall wells of a single plate, and therefore a constant volume of polymersolution per reaction (see methods below).

[0249] The efficiency of in vitro transfection using cationic polymerssuch as poly(ethylene imine) is very sensitive to the ratio of polymerto DNA present during complex formation (Gebhart et al. Journal ofControlled Release 73:401 (2001); incorporated herein by reference). N:Prations of 10:1, 20:1, and 40:1 were selected for our initial screensbased on previous experience with these types of polymers. Using ourhigh-throughput system, we screened all 2350 polymers at these threeratios. Transfection values at the best condition for each polymer weretabulated into a histogram (FIG. 13). These results were compared tothree controls: naked DNA (no transfection agent), poly(ethylene imine)(PEI), and Lipofectamine 2000. The low, residual levels of DMSO presentin the transfection solutions did not affect transfection efficiency ofeither naked DNA or PEI. Thirty-three of the 2350 polymers were found tobe as good or better than PEI in this unoptimized system.

[0250] Since cationic polymer transfections tend to be sensitive topolymer:DNA ratio, we decided to optimize transfection conditions withthe best polymers from our preliminary screen. Using the results aboveas a rough guide, the transfection condition for the best 93 polymerswere optimized by testing them at N:P ratios above and below the optimaltransfection conditions identified in the broad based screen. In orderto develop a high-throughput optimization system, these were testedusing an N:P ratio multiplier system to simplify simultaneous testingand plate-to-plate transfer. Starting with the best N:P ratio identifiedin the preliminary screen, the polymers were retested at six N:P ratiosequal to the best ratio times 0.5, 0.75, 1.0, 1.25, 1.5, and 1.75, intriplicate. For example, if the optimal ratio identified in the screenfor a given polymer was 20: 1, then that polymer was rescreened intriplicate at N:P ratios of 10:1, 15:1, 20:1, 25:1, 30:1, and 35:1. Theaverage transfection efficiencies with standard deviation from the bestcondition for the best 50 polymers are shown in FIG. 14, along withcontrol data. In this experiment, 46 polymers were identified thattransfect as good or better than PEI. All 93 of these polymers were alsotested for their ability to bind DNA using agarose gel electrophoresis(FIG. 15). Interestingly, while almost all of the polymers bind DNA asexpected, two polymers that transfect at high levels do not: M17 andKK89 (FIG. 14).

[0251] To further examine the transfection properties of these polymers,ten high transfecting polymers were tested for their ability to deliverthe green fluorescent protein plasmid, pCMV-eGFP. Unlike pCMV-luc,pCMV-eGFP provides information concerning what percentage of cells istransfected. High levels of transfection were observed for all 10polymers, and two of the best are shown in FIG. 16.

[0252] The “hits” identified in the above assays reveal a surprisinglydiverse and unexpected collection of polymers. Particularly surpising isthe large collection of hydrophobic, D-monomer-based polymers. In fact,the diacrylate monomers used to make the best performing 50 polymers arealmost always hydrophobic. Further analysis reveals two more commonfeatures of the effective polymers: 1) twelve of the 26 polymers thatare better than the best conventional reagent, Lipofectamine 2000, havemono- an di-alcohol side groups, and 2) linear, bis(secondary amines)are also prevelant in the hit structures. Also surprising was theidentification of two polymers that transfect at high levels but do notappear to bind DNA (KK89 and M17). Both are also insoluble at pH 5 andpH 7. Their ability to facilitate DNA uptake may be due topermeabilization of the cellular membrane.

[0253] Also important for the function of gene delivery polymers islength (Remy et al. Advanced Drug Delivery Reviews 30:85 (1998);Schaffer et al. Biotechnol Bioeng 67:598 (2000); each of which isincorporated herein by reference). Using these results as a framework, arange of polymer lengths for each hit polymer may be prepared bycarefully varying relative monomer concentrations. Evidence shows that(1) like PEI, poly(beta-amino ester) length is important in the genedelivery proficiency of these polymers, and (2) that the hits identifiedhere can be resynthesized using conventional methods and still deliverDNA effectively.

[0254] Experimental Protocols

[0255] Polymer synthesis. Monomers were purchased from Aldrich(Milwaukee, Wis.), TCI (Portland, Oreg.), Pfaltz & Bauer (Waterbury,Conn.), Matrix Scientific (Columbia, S.C.), Acros-Fisher (Pittsburg,Pa.), Scientific Polymer (Ontario, N.Y.), Polysciences (Warrington,Pa.), and Dajac monomer-polymer (Feasterville, Pa.). These weredissolved in DMSO (Aldrich) to a final concentration of 1.6 M. Allpossible pair wise combinations amine and diacrylate monomers were addedin 150 μl aliquots to each well of 2 ml 96 well polypropylenemasterblock deep well plates (Griener America, Longwood, Fla.). Theplates were sealed with aluminum foil, and incubated at 56° C. whilerotating on an orbital shaker. After 5 days, 1 ml of DMSO was added toeach well, and the plates were resealed and stored frozen at 4° C. untilready to be used. Transfection experiments. 14,000 cos-7 cells (ATCC,Manassas, Va.) were seeded into each well of a solid white or clear 96well plate (Corning-Costar, Kennebunk, Me.) and allowed to attachedovernight in growth medium, composed of: 500 ml phenol red minus DMEM,50 ml heat inactivated FBS, 5 ml penicillin/streptomycin (Invitrogen,Carlsbad, Calif.). Each well of a master block 96-well plate was filledwith 1 ml of 25 mM sodium acetate pH 5. To this, 40 μl, 20 μl, or 10 μlof the polymer/DMSO solution was added. 25 μl of the diluted polymer wasadded to 25 μl of 60 μg/ml pCMV-luc DNA (Promega, Madison, Wis.) orpEGFP-N1 (Invitrogen) in a half volume 96 well plate. These wereincubated for 10 minutes, and then 30 μl of the polymer-DNA solution wasadded to 200 μl of Optimem with sodium bicarbonate (Invitrogen) in 96well polystyrene plates. The medium was removed from the cells using a12-channel wand (V & P Scientific, San Diego, Calif.) after which 150 μlof the optimem-polymer-DNA solution was immediately added. Complexeswere incubated with the cells for 1 hour and then removed using the12-channel wand and replaced with 105 μl of growth medium. Cells wereallowed to grow for three days at 37° C., 5% CO₂ and then analyzed forluminescence (pCMV-luc) or fluorescence (pEGFP-N1). Control experimentswere performed by as described above, but using poly(ethylene imine) MW25,000 (Aldrich) replacing synthesized polymer, and at polymer:DNAweight ratios of 0.5:1, 0.75:1, 1:1, 1.25:1, 1.5:1, and 2:1.Lipofectamine 2000 (Invitrogen) transfections were performed asdescribed by the vendor, except that complexes were removed after 1hour.

[0256] Luminescence was analyzed using bright-glo assay kits (Promega).Briefly, 100 μl of bright-glo solution was added to each well of themicrotiter plate containing media and cells. Luminescence was measuredusing a Mithras Luminometer (Berthold, Oak Ridge, Tenn.). In some cases,a neutral density filter (Chroma, Brattleboro, Vt.) was used to preventsaturation of the luminometer. A standard curve for Luciferase wasgenerated by titration of Luciferase enzyme (Promega) into growth mediain white microtiter plates. eGFP expression was examined using a ZeissAciovert 200 inverted microscope.

[0257] Agarose gel electrophoresis DNA-binding assays were done at a N:Pratio of 40: 1, as previously described (Lynn et al. Journal of theAmerican Chemical Society 123:8155 (2001): incorporated herein byreference). All liquid handling was performed using an EDR384S/96S robot(Labcyte, Union City, Calif.) or a 12 channel micropippettor (ThermoLabsystems, Vantaa, Finland) in a laminar flow hood.

Example 5 Synthesis of Poly(beta-amino esters) Optimized for HighlyEffective Gene Delivery

[0258] The effect of molecular weight, polymer/DNA ratio, and chainend-group on the transfection properties of two unique poly(β-aminoester) structures was determined. These factors can have a dramaticeffect on gene delivery function. Using high throughput screeningmethods, poly(β-amino esters) that transfect better than PEI andLipofectamine 2000 (two of the best commercially available transfectionreagents) have been discovered.

[0259] Materials and Methods

[0260] Polymer Synthesis. Poly-1 and Poly-2 polymers were synthesized byadding 1,4-butanediol diacrylate (99+%) and 1,6-hexanediol diacrylate(99%), respectively, to 1-amino butanol (98%). These monomers werepurchased from Alfa Aesar (Ward Hill, Mass.). Twelve versions each ofPoly-1 and Poly-2 were generated by varying the amine/diacrylatestoichiometric ratio. To synthesize each of the 24 unique polymers, 400mg of 1-amino butanol was weighed into an 8 mL sample vial withTelfon-lined screw cap. Next, the appropriate amount of diacrylate wasadded to the vial to yield a stoichiometric ratio between 1.4 and 0.6. Asmall Telfon-coated stir bar was then put in each vial. The vials werecapped tightly and placed on a multi-position magnetic stir-plateresiding in an oven maintained at 100° C. After a reaction time of 5 hr,the vials were removed from the oven and stored at 4° C. All polymerswere analyzed by GPC. Gel Permeation Chromatography (GPC). GPC wasperformed using a Hewlett Packard 1100 Series isocratic pump, a RheodyneModel 7125 injector with a 100-μL injection loop, and a Phenogel MXLcolumn (5μ mixed, 300×7.5 mm, Phenomenex, Torrance, Calif.). 70% THF/30%DMSO (v/v)+0.1 M piperidine was used as the eluent at a flow rate of 1.0mL/min. Data was collected using an Optilab DSP interferometricrefractometer (Wyatt Technology, Santa Barbara, Calif.) and processedusing the TriSEC GPC software package (Viscotek Corporation, Houston,Tex.). The molecular weights and polydispersities of the polymers weredetermined relative to monodisperse polystyrene standards.

[0261] Luciferase Transfection Assays. COS-7 cells (ATCC, Manassas, Va.)were seeded (14,000 cells/well) into each well of an opaque white96-well plate (Corning-Costar, Kennebunk, Me.) and allowed to attachovernight in growth medium. Growth medium was composed of 90% phenolred-free DMEM, 10% fetal bovine serum, 100 units/mL penicillin, 100μg/mL streptomycin (Invitrogen, Carlsbad, Calif.). To facilitatehandling, polymers stock solutions (100 mg/mL) were prepared in DMSOsolvent. A small residual amount of DMSO in the transfection mixturedoes not affect transfection efficiency and does not result in anyobservable cytotoxicity. Working dilutions of each polymer were prepared(at concentrations necessary to yield the different polymer/DNA weightratios) in 25 mM sodium acetate buffer (pH 5). 25 μl of the dilutedpolymer was added to 25 μl of 60 μg/ml pCMV-Luc DNA (ElimBiopharmaceuticals, South San Francisco, Calif.) in a well of a 96-wellplate. The mixtures were inculated for 10 minutes to allow for complexformation, and then 30 μl of the each of the polymer-DNA solutions wereadded to 200 μl of Opti-MEM with sodium bicarbonate (Invitrogen) in96-well polystyrene plates. The growth medium was removed from the cellsusing a 12-channel aspirating wand (V&P Scientific, San Diego, Calif.)after which 150 μl of the Opti-MEM-polymer-DNA solution was immediatelyadded. Complexes were incubated with the cells for 1 hr and then removedusing the 12-channel wand and replaced with 105 μl of growth medium.Cells were allowed to grow for three days at 37° C., 5% CO₂ and werethen analyzed for luciferase expression. Control experiments were alsoperformed with PEI (MW=25,000, Sigma-Aldrich) and Lipofectamine 2000(Invitrogen). PEI transfections were performed as described above, butusing polymer:DNA weight ratios of 1:1. Lipofectamine 2000 transfectionswere performed as described by the vendor, except that complexes wereremoved after 1 hour.

[0262] Luciferase expression was analyzed using Bright-Glo assay kits(Promega). Briefly, 100 μl of Bright-Glo solution was added to each wellof the 96-well plate containing media and cells. Luminescence wasmeasured using a Mithras Luminometer (Berthold, Oak Ridge, Tenn.). A 1%neutral density filter (Chroma, Brattleboro, Vt.) was used to preventsaturation of the luminometer. A standard curve for Luciferase wasgenerated by titration of Luciferase enzyme (Promega) into growth mediain an opaque white 96-well plate.

[0263] Measurement of Cytotoxicity. Cytotoxicity assays were performedin the same manner as the luciferase transfection experiments with thefollowing exception. Instead of assaying for luciferase expression after3 days, cells were assayed for metabolic activity using the MTT CellProliferation Assay kit (ATCC) after 1 day. 10 μL of MTT Reagent wasadded to each well. After 2 hr incubation at 37° C., 100 μL of DetergentReagent was added to each well. The plate was then left in the dark atroom temperature for 4 hr. Optical absorbance was measured at 570 nmusing a SpectaMax 190 microplate reader (Molecular Devices, Sunnyvale,Calif.) and converted to % viability relative to control (untreated)cells.

[0264] Cellular Uptake Experiments. Uptake experiments were done aspreviously described, with the exception that a 12-well plate format wasused instead of a 6-well plate format (Akinc, A., et al., Parallelsynthesis and biophysical characterization of a degradable polymerlibrary of gene delivery. J. Am. Chem. Soc., 2003; incorporated hereinby reference). COS-7 cells were seeded at a concentration of 1.5×10⁵cells/well and grown for 24 hours prior to performing the uptakeexperiments. Preparation of polymer/DNA complexes was done in the samemanner as in the luciferase transfection experiments, the onlydifferences being an increase in scale (2.5 μg DNA per well of 12-wellplate as opposed to 600 ng DNA per well of 96-well plate) and the use ofCy5-labeled plasmid instead of pCMV-Luc (Akinc, A. and R. Langer,Measuring the pH environment of DNA delivered using nonviral vectors:Implications for lysosomal trafficking. Biotechnol. Bioeng., 2002.78(5): p. 503-8; incorporated herein by reference). As in thetransfection experiments, complexes were incubated with cells for 1 hrto allow for cellular uptake by endocytosis. The relative level ofcellular uptake was quantified using a flow cytometer to measure thefluorescence of cells loaded with Cy5-labeled plasmid.

[0265] GFP Transfections. GFP transfections were carried in COS-7 (greenmonkey kidney), NIH 3T3 (murine fibroblast), HepG2 (humanhepatocarcinoma), and CHO (Chinese Hamster Ovary) cell lines. All celllines were obtained from ATCC (Manassas, Va.) and maintained in DMEMcontaining 10% fetal bovine serum, 100 units/mL penicillin, 100 μg/mLstreptomycin at 37° C. in 5% CO₂ atmosphere. Cells were seeded on 6-wellplates and grown to roughly 80-90% confluence prior to performing thetransfection experiments. Polymer/DNA complexes were prepared asdescribed above using the pEGFP-N1 plasmid (Clontech, Palo Alto, Calif.)(5 μg/well). Complexes were diluted in 1 mL Opti-MEM and added to thewells for 1 hr. The complexes were then removed and fresh growth mediawas added to the wells. After 2 days, cells were harvested and analyzedfor GFP expression by flow cytometry. Propidium iodide staining was usedto exclude dead cells from the analysis.

[0266] Flow Cytometry. Flow cytometry was performed with a FACSCalibur(Becton Dickinson) equipped with an argon ion laser capable of excitingGFP (488 nm excitation) and a red diode laser capable of exciting Cy5(635 nm excitation). The emission of GFP was filtered using a 530 nmband pass filter and the emission of Cy5 was filtered using a 650 longpass filter. The cells were appropriately gated by forward and sidescatter and 30,000 events per sample were collected.

[0267] Results and Discussion

[0268] Polymer Synthesis. As previously described (Lynn, D. M. and R.Langer, Degradable poly(β-amino esters): synthesis, characterization,and self-assembly with plasmid DNA. J. Am. Chem. Soc., 2000.122(44): p.10761-10768; incorporated herein by reference), the synthesis ofpoly(β-amino esters) proceeds via the conjugate addition of amines toacrylate groups. Because the reaction is a step polymerization, a broad,statistical distribution of chain lengths is obtained, with averagemolecular weight and chain end-groups controlled by monomerstoichiometry (Flory, P., in Principles of Polymer Chemistry. 1953,Cornell University Press: Ithaca, N.Y. p. 40-46, 318-323; Odian, G.,Step Polymerizaton, in Principles of Polymerization. 1991, John Wiley &Sons, Inc.: New York. p. 73-89; each of which is incorporated herein byreference). Molecular weight increases as the ratio of monomers nearsstoichiometric equivalence, and an excess of amine or diacrylate monomerresults in amine- or acrylate-terminated chains, respectively. For thisclass of polymers, precise control of stoichiometry is essential forcontrolling polymer molecular weight. While monomer stoichiometry is themost important factor affecting chain length, consideration should alsobe given to competing side reactions that can impact the molecularweight and structure of polymer products. In particular, intramolecularcyclization reactions, where an amine on one end of the growing polymerchain reacts with an acrylate on the other end, can limit obtainedmolecular weights (Odian, G., Step Polymerizaton, in Principles ofPolymerization. 1991, John Wiley & Sons, Inc.: New York. p. 73-89;incorporated herein by reference). These cyclic chains may also haveproperties that differ from those of their linear counterparts.

[0269] In this work, we have modified the previously reportedpolymerization procedure in order to better control monomerstoichiometry and to minimize cyclization reactions. First, the scale ofsynthesis was increased from roughly 0.5 g to 1 g to allow for controlof stoichiometry within 1%. Further improvement in accuracy is limitedby the purity (98-99%) of the commercially available monomers used.Second, all monomers were weighed into vials instead of being dispensedvolumetrically. Discrepancies between actual and reported monomerdensities were found to be non-negligible in some cases, leading toinaccuracies in dispensed mass. Third, polymerizations were performed inthe absence of solvent to maximize monomer concentration, thus favoringthe intermolecular addition reaction over the intramolecular cyclizationreaction. Eliminating the solvent also provides the added benefits ofincreasing the reaction rate and obviating the solvent removal step.Finally, since the previously employed methylene chloride solvent wasnot used, the reaction temperature could be increased from 45° C. to100° C. Increasing temperature resulted in an increased reaction rateand a decrease in the viscosity of the reacting mixture, helping tooffset the higher viscosity of the solvent-free system. The combinedeffect of increased monomer concentration and reaction temperatureresulted in a decrease in reaction time from roughly 5 days to 5 hours.

[0270] We synthesized polymers Poly-1 and Poly-2 by adding1,4-butanediol diacrylate and 1,6-hexanediol diacrylate, respectively,to 1-amino butanol. Twelve unique versions of each polymer weresynthesized by varying amine/diacrylate mole ratios between 0.6 and 1.4.

[0271] For both sets of polymers (Poly-1 and Poly-2), 7 of the 12 hadamine/diacrylate ratios>1, resulting in amine-terminated polymers, and 5of the 12 had amine/diacrylate ratios<1, resulting inacrylate-terminated polymers. After 5 hr reaction at 100° C., polymerswere obtained as clear, slightly yellowish, viscous liquids. Thepolymers had observable differences in viscosity, corresponding todifferences in molecular weight. Polymers were analyzed by organic phasegel permeation chromatography (GPC) employing 70% THF/30% DMSO (v/v)+0.1M piperidine eluent. Polymer molecular weights (M_(w)) ranged from 3350(Poly-1, amine/diacrylate=1.38) to 18,000 (Poly-1,amine/diacrylate=0.95), relative to polystyrene standards (FIG. 16).Molecular weight distributions were monomodal with polydispersityindices (PDIs) ranging from 1.55 to 2.20.

[0272] Luciferase Transfection Results. Transfection experiments wereperformed with all 24 synthesized polymers (12 each of Poly-1 andPoly-2) at 9 different polymer/DNA ratios to determine the impact ofmolecular weight, polymer/DNA ratio, and chain end-group on transfectionefficiency (FIGS. 17 and 18). As a model system, we used the COS-7 cellline and a plasmid coding for the firefly luciferase reporter gene(pCMV-Luc) (600 ng/well). To facilitate performance of the nearly 1000transfections (data obtained in quadruplicate), experiments were done in96-well plate format. Reporter protein expression levels were determinedusing a commercially available luciferase assay kit and a 96-wellluminescence plate reader.

[0273] The data displayed in FIGS. 17 and 18 demonstrate that polymermolecular weight, polymer/DNA ratio, and chain end-group impact thetransfection properties of both Poly-1 and Poly-2 polymers. Onestriking, and somewhat unexpected, result was that none of theacrylate-terminated polymers mediated appreciable levels of transfectionunder any of the evaluated conditions. This result may be more broadlyapplicable for poly(β-amino esters), as we have yet to synthesize apolymer, using an excess of diacrylate monomer, that mediatesappreciable reporter gene expression at any of the polymer/DNA ratios wehave employed. These findings suggest that perhaps only amine-terminatedpoly(β-amino esters) are suitable for use as gene delivery vehicles. Incontrast, there were distinct regions of transfection activity in theMWpolymer/DNA space for amine-terminated versions of both Poly-1 andPoly-2 (FIGS. 17-A and 18-A). Maximal reporter gene expression levels of60 ng luc/well and 26 ng luc/well were achieved using Poly-1(M_(w)=13,100) and Poly-2 (M_(w)=13,400), respectively. These resultscompare quite favorably with PEI (polymer/DNA=1:1 w/w), which mediatedan expression level of 6 ng luc/well (data not shown) under the sameconditions.

[0274] While the highest levels of transfection occurred using thehigher molecular weight versions of both polymer structures, the optimalpolymer/DNA ratios for these polymers were markedly different(polymer/DNA=150 for Poly-1, polymer/DNA=30 for Poly-2). Thetransfection results we have obtained for Poly-1 and Poly-2 highlightthe importance of optimizing polymer molecular weight and polymer/DNAratio, and the importance of controlling chain end-groups. Further, thefact that two such similar polymer structures, differing by only twocarbons in the repeat unit, have such different optimal transfectionparameters emphasizes the need to perform these optimizations for eachunique polymer structure. To improve our understanding of the obtainedtransfection results, we chose to study two important deliverycharacteristics that directly impact transgene expression, cytotoxicityand the ability to enter cells via endocytosis (Wiethoff, C. M. and C.R. Middaugh, Barriers to nonviral gene delivery. Journal ofPharmaceutical Sciences, 2003. 92(2): p. 203-217; incorporated herein byreference).

[0275] Cytotoxicity. We evaluated the cytotoxicities of the variouspolymer/DNA complexes using a standard MTT/thiazolyl blue dye reductionassay. The experiments were performed exactly as the transfectionexperiments described above, except that instead of assaying forreporter gene expression on day 3, the MTT assay was performed after day1 (see Materials and Methods). We initially hypothesized that the lackof transfection activity observed for acrylate-terminated polymers mayhave been due to the cytotoxicity of the acrylate end-groups. FIGS. 19-Band 20-B do indicate that high concentrations of acrylate are cytotoxic,as viability is seen to decrease with increasing polymer/DNA ratio anddecreasing polymer M_(w) (lower M_(w) corresponds to a higherconcentration of end-groups). However, cytotoxicity of acrylates doesnot sufficiently explain the lack of transfection activity at lowerpolymer/DNA ratios or higher molecular weights. Data shown in FIG. 19-Ademonstrates that cytotoxicity is not a limiting factor for Poly-1vectors, since cells remain viable even at the highest polymer/DNAratios. On the other hand, the data displayed in FIG. 20-A suggests thatcytotoxicity is a major factor limiting the transfection efficiency ofPoly-2 vectors, especially for the lower molecular weight polymers. Thisresult may explain why transfection activity is non-existent ordecreasing for Poly-2 vectors at polymer/DNA>30 (see FIG. 18-A).

[0276] Cellular Uptake. The ability of polymer/DNA complexes to be takenup by cells was evaluated using a previously described flowcytometry-based technique to measure the fluorescence ofvector-delivered DNA (Akinc, A., et al., Parallel synthesis andbiophysical characterization of a degradable polymer library of genedelivery. J. Am. Chem. Soc., 2003; incorporated herein by reference).Briefly, polymer/DNA complexes were prepared using plasmid DNAcovalently labeled with the fluorescent dye Cy5. To allow for comparisonof the cellular uptake data with the gene expression data outlinedabove, complexes were formed at the same polymer/DNA ratios and in thesame manner as in the transfection experiments. Labeled complexes wereincubated with COS-7 cells for 1 hr at 37° C. to allow for uptake. Therelative level of particle uptake was then quantified by measuring thefluorescence of cells loaded with Cy5-labeled DNA. The results of theseuptake experiments are summarized in FIGS. 21 and 22. Data shown in FIG.21-B and 22-B suggest that the lack of transfection activity for theacrylate-terminated polymers is not due to cytotoxicity, as initiallythought, but rather to an inability to enter the cell. Similarly, Poly-1complexes are severely uptake-limited at all but the highest polymer/DNAratios (FIG. 21-A). While this data doesn't correlate exactly with thetransfection results obtained for Poly-1, it is consistent with the factthat transfection activity is not observed until very high polymer/DNAratios are employed. Poly-2 complexes show no appreciable cellularuptake at polymer/DNA ratios<30 and increasing levels of uptake aspolymer/DNA ratios increase beyond 30 (FIG. 22-A). This result, combinedwith the above cytotoxicity results, helps to explain the transfectionactivity of Poly-2 complexes. At polymer/DNA ratios less than 30,complexes do not effectively enter the cell, but as polymer/DNA ratiosincrease much beyond 30, cytotoxicity begins to limit transfectionefficiency, resulting in optimal transfection activity nearpolymer/DNA=30.

[0277] Where endocytosis is the main route of cellular entry, theeffective formation of nanometer-scale polymer/DNA complexes is onerequirement for achieving high levels of cellular uptake (De Smedt, S.C., J. Demeester, and W. E. Hennink, Cationic polymer based genedelivery systems. Pharmaceutical Research, 2000. 17(2): p. 113-126;Prabha, S., et al., Size-dependency of nanoparticle-mediated genetransfection: studies with fractionated nanoparticles. InternationalJournal of Pharmaceutics, 2002. 244(1-2): p. 105-115; each of which isincorporated herein by reference). The poor uptake levels observed formany of the polymer/DNA complexes may have been attributable to theunsuccessful formation of stable, nanoscale complexes. Unfortunately,the poor neutral pH solubility of the polymers prevented making dynamiclight scattering measurements of complex size using the transfectionmedium (Opti-MEM reduced serum media, pH 7.2) as the diluent. Theobtained readings were attributable to polymer precipitates, which werein some cases visible as turbidity in solutions of polymer in Opti-MEM.However, we were able to measure the effective diameters of complexesusing 25 mM sodium acetate (pH 5) as the sample diluent. While this datacannot shed light on the size or stability of complexes in thetransfection medium, it can indicate whether complexes were successfullyformed prior to addition to the transfection medium. Specifically, wefound that the lower molecular weight versions (M_(w)<10,700) of Poly-1were unable to form nanoscale complexes, even in acetate buffer. In allother cases we found that nanometer-sized polymer/DNA complexes wereformed. While these results may explain the poor uptake levelsassociated with low molecular weight versions of Poly-1, they do notsatisfactorily explain the low uptake activity of theacrylate-terminated polymers or the dependence of polymer/DNA ratio oncellular uptake. Although particle size and stability are importantfactors impacting cellular uptake, it is likely that other, yetunidentified, factors must also be considered in order to provide a morecomplete explanation of the obtained cellular uptake results.

[0278] Enhancement of Transfection Using a Co-Complexing Agent. BothPoly-1 and Poly-2 require relatively high polymer/DNA weight ratios toachieve high levels of gene transfer. One explanation may be that,compared to other polymers often used to compact DNA (e.g., polylysine(PLL) and PEI), these polymers have relatively low nitrogen densities.Poly-1 has a molecular weight per nitrogen atom (MW/N) of 301, andPoly-2 has a MW/N of 329. By comparison, for PLL and PEI, these figuresare roughly 65 and 43, respectively. It might be possible to reduce theamount of Poly-1 or Poly-2 necessary to achieve high levels oftransfection by incorporating a small amount of co-complexing agent.This approach could be especially beneficial for Poly-2 vectors, sincecytotoxicity appears to be an important limitation for these vectors. Totest this hypothesis, PLL and PEI were used as model co-complexingagents. We focused our attention on the most promising member in each ofthe Poly-1 (amine-terminated, M_(w)=13,100) and Poly-2(amine-terminated, M_(w)=13,400) sets of polymers. The data displayed inFIGS. 23 and 24 indicate that a significant reduction in polymer couldbe achieved, while maintaining high levels of transfection efficiency,through the use of PLL or PEI as co-complexing agents. In some cases,significant enhancement of transfection activity was realized. Asexpected, this co-complexation approach was particularly beneficial forPoly-2 vectors. This work, and prior work (Wagner, E., et al., Influenzavirus hemagglutinin HA-2 N-terminal fusogenic peptides augment genetransfer by transferrin-polylysine-DNA complexes: toward a syntheticvirus-like gene-transfer vehicle. Proc. Natl. Acad. Sci. U.S.A., 1992.89(17): p. 7934-8; Fritz, J. D., et al., Gene transfer into mammaliancells using histone-condensed plasmid DNA. Hum Gene Ther, 1996. 7(12):p. 1395-404; Pack, D. W., D. Putnam, and R. Langer, Design ofimidazole-containing endosomolytic biopolymers for gene delivery.Biotechnol. Bioeng., 2000. 67(2): p. 217-23; Lim, Y. B., et al.,Self-Assembled Ternary Complex of Cationic Dendrimer, Cucurbituril, andDNA: Noncovalent Strategy in Developing a Gene Delivery Carrier.Bioconjug Chem, 2002. 13(6): p. 1181-5; each of which is incorporatedherein by reference), demonstrates that the blending of polymers withcomplementary gene transfer characteristics can in some cases producemore effective gene delivery reagents.

[0279] GFP Transfections

[0280] To further evaluate the transfection properties of the Poly-1/PLLand Poly-2/PLL blended reagents, we performed transfection experimentsusing a reporter plasmid coding for green fluorescent protein(pCMV-EGFP). Though the luciferase and GFP transfection assay systemsboth measure transfection activity, they provide different types ofinformation. The luciferase system quantifies the total amount ofexogenous luciferase protein produced by all the cells in a given well,providing a measure of cumulative transfection activity. In contrast,the GFP system can be used to quantify the percentage of cells that havebeen transfected, providing a cell-by-cell measure of transfectionactivity. Both systems are useful and offer complementary informationregarding the transfection properties of a given gene delivery system.

[0281] GFP transfections were performed in a similar manner as theluciferase experiments, but were scaled up to 6-well plate format (5 μgplasmid/well). COS-7 cells were transfected using Poly-1/PLL(Poly-1:PLL:DNA=60:0.1:1 w/wlw) and Poly-2/PLL (Poly-2:PLL:DNA=15:0.4:1w/w/w). Lipofectamine 2000 (μL reagent: μg DNA=1:1), PEI (PEI:DNA=1:1w/w, N/P˜8), and naked DNA were used as controls in the experiment.After 1 hr incubation with cells, vectors were removed and fresh growthmedia was added. Two days later GFP expression was assayed using a flowcytometer. Nearly all cells transfected with Poly-1/PLL were positivefor GFP expression (FIGS. 25 and 26). Experiments indicated thatPoly-2/PLL vectors were less effective, resulting in roughly 25%positive cells. Positive controls Lipofectamine 2000 and PEI were alsoable to mediate effective transfection of COS-7 cells under theconditions employed. Although Lipofectamine 2000 and PEI transfectionsresulted in nearly the same percentage of GFP-positive cells asPoly-1/PLL, the fluorescence level of GFP-positive cells was higher forPoly-1/PLL (mean fluorescence=6033) than that of both Lipofectamine 2000(mean fluorescence=5453) and PEI (mean fluorescence=2882). Multiplyingthe percentage of positive cells by the mean fluorescence level ofpositive cells provides a measure of aggregate expression for the sampleand should, in theory, better correlate with the results of luciferasegene expression experiments. Quantifying total GFP expression in thismanner indicates that the highest expression level is achieved byPoly-1/PLL, followed by Lipofectamine 2000 and PEI. This result is ingeneral agreement with the luciferase expression results.

[0282] Experiments have shown that Poly-I/PLL (Poly-1:PLL:DNA=60:0.1:1w/w/w) is a highly effective vector for transfecting COS-7 cells. Theability of this vector to mediate transfection in three other commonlyused cell lines (CHO, NIH 3T3, and HepG2) was also investigated. It isvery likely that each of these cell lines have optimal transfectionconditions that differ from those used to transfect COS-7 cells;however, as a preliminary evaluation of the ability to transfectmultiple cell lines, transfections were performed in the same manner andunder the same conditions as the COS-7 transfections. Results indicatethat Poly-1/PLL (Poly1:PLL:DNA=60:0.1:1 w/w/w) is able to successfullytransfect CHO, NIH 3T3, and HepG2 cells, though not as effectively asCOS-7 cells (FIG. 27). This is not too surprising since the vector usedwas optimized by screening for gene transfer in COS-7 cells.Optimization of vector composition and transfection conditions specificfor each cell type would be expected to result in even highertransfection levels.

SUMMARY

[0283] In this work, the role of polymer molecular weight, polymer chainend-group, and polymer/DNA ratio on a number of important gene transferproperties has been investigated. All three factors were found to have asignificant impact on gene transfer, highlighting the benefit ofcarefully controlling and optimizing these parameters. In addition, theincorporation of a small amount of PLL, used to aid complexation,further enhances gene transfer. Through these approaches degradablepoly(beta-amino esters)-based vectors that rival some of the bestavailable non-viral vectors for in vitro gene transfer.

Example 6 Further Studies of Selected poly(beta-amino esters)

[0284] To further characterize and synthesize some of thepoly(beta-amino esters) identified in previous screens, a twenty-onepolymers were re-synthesized at various ratios of amine monomer toacrylate monomer. The resulting polymers were characterized by gelpermeation chromatography to determine the molecular weight andpolydispersitie of each polymer. Each of the polymer was then tested forits ability to transfect cells.

[0285] Polymer Synthesis. The polymers were synthesized as described inExample 5. Several versions of each polymer were created by varying theamin/diacrylated stoichiometric ratio. For example, C36-1 corresponds tothe stoichiometric ratio of 1.4, and C36-12 to 0.6, with all theintermediates given in the table below: Amine:Acrylate Version of C36Stoichiometric Ratio C36-1 1.4 C36-2 1.3 C36-3 1.2 C36-4 1.1 C36-5 1.05C36-6 1.025 C36-7 1.0 C36-8 0.975 C36-9 0.950 C36-10 0.9 C36-11 0.8C36-12 0.6

[0286] The polymers were typically prepared in glass vials with nosolvent at 100° C. for 5 hours. In some syntheses, the polymerization at100° C. yielded highly cross-linked polymers when certain monomers suchas amine 94 were used; therefore, the polymerization reactions wererepeated at 50° C. with 2 mL of DMSO added to avoid cross-linking.

[0287] The resulting polymers were analyzed by GPC as described inExample 5. The molecular weights and polydispersities of each of thepolymers is shown in the table below: Polymer M_(w) M_(n) PolydispersityF28-1 5540 2210 2.50678733 F28-2 6150 2460 2.5 F28-3 8310 29202.845890411 F28-4 11600 3660 3.169398907 F28-5 16800 4360 3.853211009F28-6 16100 4850 3.319587629 F28-7 18000 5040 3.571428571 F28-8 182005710 3.187390543 F28-9 22300 7880 2.829949239 F28-10 23700 87802.699316629 F28-11 12100 5660 2.137809187 F28-12 4850 2920 1.660958904C36-1 7080 3270 2.165137615 C36-2 5100 2640 1.931818182 C36-3 21200 80902.620519159 C36-4 20500 6710 3.05514158 C36-5 112200 33200 3.379518072C36-6 21700 6890 3.149492017 C36-7 36800 15700 2.343949045 C36-8 3570012600 2.833333333 C36-9 35200 15100 2.331125828 C36-10 22500 98902.275025278 C36-11 26000 6060 4.290429043 D60-1 1890 1400 1.35 D60-22050 1520 1.348684211 D60-3 2670 1720 1.552325581 D60-4 3930 22101.778280543 D60-5 5130 2710 1.89298893 D60-6 5260 2800 1.878571429 D60-71130 1090 1.036697248 D60-8 1840 1510 1.218543046 D60-9 6680 34401.941860465 D60-10 8710 4410 1.975056689 D60-11 9680 4410 2.195011338D60-12 7450 3470 2.146974063 D61-1 1710 1410 1.212765957 D61-2 2600 17901.452513966 D61-3 3680 2280 1.614035088 D61-4 4630 2550 1.815686275D61-5 NA NA NA D61-6 NA NA NA D61-7 6110 3250 1.88 D61-8 6410 31902.009404389 D61-9 6790 3440 1.973837209 D61-10 8900 4350 2.045977011D61-11 10700 4600 2.326086957 D61-12 6760 2900 2.331034483 F32-1 103003260 3.159509202 F32-2 11100 3490 3.180515759 F32-3 16600 48203.443983402 F32-4 17300 5390 3.209647495 F32-5 18600 5830 3.190394511F32-6 26200 8290 3.160434258 C32-1 6670 2810 2.37366548 C32-2 18100 56803.186619718 C32-3 19300 6060 3.184818482 C32-4 25600 9100 2.813186813C32-5 25000 7860 3.180661578 C32-6 25700 8440 3.045023697 C86-1 142002900 4.896551724 C86-2 21000 2900 7.24137931 C86-3 27500 45905.991285403 U94-1 10700 3530 3.031161473 U94-2 NA NA NA U94-3 NA NA NAF32-1 10300 3260 3.159509202 F32-2 11100 3490 3.180515759 F32-3 166004820 3.443983402 F32-4 17300 5390 3.209647495 F32-5 25000 78603.180661578 F32-6 26200 8290 3.160434258 C32-1 6670 2810 2.37366548C32-2 18100 5680 3.186619718 C32-3 19300 6060 3.184818482 C32-4 256009100 2.813186813 C32-5 25000 7860 3.180661578 C32-6 25700 84403.045023697 U86-1 Unusual NA NA U86-2 Unusual NA NA U86-3 Unusual NA NAU86-4 Unusual NA NA U85-5 Unusual NA NA JJ32-1 9730 4010 2.426433915JJ32-2 12100 4580 2.641921397 JJ32-3 19400 6510 2.980030722 JJ32-4 2790010000 2.79 JJ32-5 32600 9720 3.353909465 JJ32-6 28900 9870 2.928064843JJ36-1 7540 3550 2.123943662 JJ36-2 143500 59600 2.407718121 JJ36-320100 7310 2.749658003 JJ36-4 30200 10200 2.960784314 JJ36-5 33900 106003.198113208 JJ36-6 36100 12500 2.888 JJ28-1 7550 3240 2.330246914 JJ28-29490 3460 2.742774566 JJ28-3 16800 5420 3.099630996 JJ28-4 23300 80902.880098888 JJ28-5 25500 7700 3.311688312 JJ28-6 32100 10900 2.944954128U28-1 7190 2580 2.786821705 U28-2 10700 3990 2.681704261 U28-3 156007300 2.136986301 U28-4 20400 9880 2.064777328 U28-5 20500 96702.119958635 U28-6 24200 13000 1.861538462 E28-1 5900 3280 1.798780488E28-2 7950 3550 2.23943662 E28-3 14300 6300 2.26984127 E28-4 6990 33202.105421687 E28-5 17400 8180 2.127139364 E28-6 19300 9030 2.137320044LL6-1 2380 1570 1.515923567 LL6-2 3350 2070 1.618357488 LL6-3 4110 23401.756410256 LL6-4 5750 3010 1.910299003 LL6-5 7810 5050 1.546534653LL6-6 6950 4190 1.658711217 LL8-1 3160 1910 1.654450262 LL8-2 3630 25601.41796875 LL8-3 5300 3520 1.505681818 LL8-4 6000 3320 1.807228916 LL8-58160 4730 1.725158562 LL8-6 7190 4650 1.546236559 U36-1 7290 33702.163204748 U36-2 11100 5000 2.22 U36-3 12600 5470 2.303473492 U36-421500 8550 2.514619883 U36-5 24700 9430 2.619300106 U36-6 31700 107002.962616822 E36-1 6030 3130 1.926517572 E36-2 8510 4040 2.106435644E36-3 12800 5730 2.233856894 E36-4 18200 7620 2.388451444 E36-5 201008050 2.49689441 E36-6 32900 10900 3.018348624 U32-1 9830 37902.593667546 U32-2 12000 4460 2.69058296 U32-3 18200 6780 2.684365782U32-4 25200 11100 2.27027027 U32-5 26500 9360 2.831196581 U32-6 2620010600 2.471698113 E32-1 7070 3310 2.135951662 E32-2 9920 41802.373205742 E32-3 14700 6080 2.417763158 E32-4 23500 9160 2.565502183E32-5 28800 10000 2.88 E32-6 26900 10300 2.611650485 C94-1 6760 31102.173633441 C94-2 10800 4190 2.577565632 C94-3 18000 5330 3.377110694C94-4 38900 6660 5.840840841 C94-5 Didn't dissolve NA NA C94-6 Didn'tdissolve NA NA D94-1 6030 2980 2.023489933 D94-2 6620 3370 1.964391691D94-3 9680 3950 2.450632911 D94-4 11500 4510 2.549889135 D94-5 137004940 2.773279352 D94-6 18800 5650 3.327433628 F94-1 5570 27402.032846715 F94-2 7670 3180 2.411949686 F94-3 12600 4230 2.978723404F94-4 20300 5160 3.934108527 F94-5 21500 5390 3.988868275 F94-6 273006310 4.326465927 JJ94-1 7750 3360 2.306547619 JJ94-2 12700 45902.766884532 JJ94-3 30500 7280 4.18956044 JJ94-4 Didn't dissolve NA NAJJ94-5 Didn't dissolve NA NA JJ94-6 Didn't dissolve NA NA F86-1 39402630 1.498098859 F86-2 5300 3190 1.661442006 F86-3 7790 4040 1.928217822F86-4 11000 5410 2.033271719 F86-5 10600 5650 1.876106195 F86-6 133006440 2.065217391 D86-1 4610 2830 1.628975265 D86-2 5570 3290 1.693009119D86-3 7120 3770 1.888594164 D86-4 8310 4440 1.871621622 D86-5 8950 47101.900212314 D86-6 10400 5010 2.075848303 U86-1 5940 3500 1.697142857U86-2 7780 4430 1.756207675 U86-3 11900 6540 1.819571865 U86-4 151007630 1.979030144 U86-5 16300 8950 1.82122905 U86-6 18100 98101.845056065 E86-1 4880 3140 1.554140127 E86-2 6300 3790 1.662269129E86-3 9780 5140 1.902723735 E86-4 12500 6350 1.968503937 E86-5 134006820 1.964809384 E86-6 15500 7280 2.129120879 JJ86-1 5460 33701.620178042 JJ86-2 6880 4080 1.68627451 JJ86-3 11900 6180 1.925566343JJ86-4 14200 7000 2.028571429 JJ86-5 20500 9090 2.255225523 JJ86-6 163007770 2.097812098 C86-1 4870 3030 1.607260726 C86-2 5720 3460 1.653179191C86-3 9970 5060 1.970355731 C86-4 14200 7000 2.028571429 C86-5 177008500 2.082352941 C86-6 17800 8500 2.094117647 C80-1 2450 17901.368715084 C80-2 3770 2370 1.5907173 C80-3 6080 3370 1.804154303 C80-47960 4310 1.846867749 C80-5 9030 4660 1.93776824 C80-6 12600 60502.082644628 E80-1 2840 2010 1.412935323 E80-2 3720 2420 1.537190083E80-3 6080 3650 1.665753425 E80-4 7210 4240 1.700471698 E80-5 7640 42901.780885781 E80-6 9000 5310 1.694915254 JJ80-1 3410 2180 1.564220183JJ80-2 4590 2890 1.588235294 JJ80-3 8430 4750 1.774736842 JJ80-4 113006560 1.722560976 JJ80-5 13200 7160 1.843575419 JJ80-6 11600 65401.773700306 U80-1 4300 2680 1.604477612 U80-2 5130 3020 1.698675497U80-3 8320 4700 1.770212766 U80-4 9130 4880 1.870901639 U80-5 11300 57501.965217391 U80-6 11200 5920 1.891891892

[0288] Luciferase Transfection Assay. As described in Example 5, COS-7cells were transfected with pCMV-Luc DNA using each of the polymers atpolymer-to-DNA ratios ranging from 10:1 to 100:1 (weight:weight).Luciferase expresseion was analysed using Bright-Glo assay kits(Promega). Luminescence was measured for each transfection, and theluminescence was used to calculate nanograms of Luciferase enzyme asdescribed in Example 5. Experiments were done in quadruplicate, and thevalues shown in the tables below are the averaged values from the fourexperiments. These data are shown below for each polymer synthesized.C36-1 C36-2 C36-3 C36-4 C36-5 C36-6 Ratio 0.168527 0.345149 0.6279920.152258 0.068355 0.094073 10 3.58467  0.12639  21.27867  2.1453880.163042 0.184298 20 4.295966 0.927605 18.84046  4.750661 0.2879891.063834 30 7.150343 1.137242 17.04771  7.529555 0.080757 0.332789 403.74705  1.180274 6.875879 9.710764 0.582186 1.963895 60 0.7056830.212297 0.560245 7.221382 5.003849 5.813189 100  C36-7 C36-8 C36-9C36-10 C36-11 C-36-12 Ratio 0.164373 0.085336 0.116502 0.042173 0.0629050.18877  10 0.134132 0.096043 0.091152 0.032851 0.032115 0.383965 200.020768 0.021203 0.0665  0.021953 0.017807 0.288102 30 0.05027 0.060731 0.017768 0.011885 0.008923 0.128469 40 0.031233 0.0488070.025626 0.012516 0.002606 0.213173 60 0.116587 0.129504 0.3324970.123413 0.058442 0.250708 100 

[0289] C86-1 C86-2 C86-3 Ratio 0.157713 0.475708 1.093272 10 0.2424810.616621 1.439904 20 0.396888 0.992601 1.758045 30 0.300173 1.2767071.901677 40

[0290] D60-1 D60-2 D60-3 D60-4 D60-5 D60-6 Ratio 0.604984 0.4438750.363271 0.260475 0.498462 0.466087 10 0.115174 0.174976 0.2506130.40783  0.587186 0.89381  20 0.138372 0.45915  0.81101  0.7731611.264634 1.438474 30 0.135287 0.506303 2.344053 1.695591 2.3023052.959638 40 0.203804 0.679718 3.908348 2.216808 3.129304 4.335511 600.233546 0.640246 0.251146 3.112999 7.65786  6.759895 100  D60-7 D60-8D60-9 D60-10 D60-11 D60-12 Ratio 0.299777 0.333863 0.434027 0.46862 0.387458 0.211083 10 0.237477 0.266398 1.211246 1.385232 1.0348920.215027 20 0.339709 0.665539 2.958346 5.607664 3.514454 0.485295 300.499842 1.216181 4.406196 6.736276 5.121445 0.444359 40 1.2973941.009228 5.951785 9.565956 7.193687 0.35831  60 5.399266 0.1358525.725666 10.45568  5.414051 0.245279 100 

[0291] D61-1 D61-2 D61-3 D61-4 D61-5 D61-6 Ratio 0.329886 0.29803 0.190101 0.142813 0.114565 0.227593 10 0.299409 0.710035 0.2955080.288845 0.247909 0.32839  20 0.155568 0.680763 0.618022 0.6516330.402721 1.831437 30 0.085824 0.620294 3.722971 4.572264 3.01027411.69027  40 0.188357 0.187979 3.970054 7.147033 10.85674  9.238981 600.019321 0.001369 0.034958 0.033062 0.202601 0.131544 100  D61-7 D61-8D61-9 D61-10 D61-11 D61-12 Ratio 0.153122 0.180646 0.1073  0.2447130.231561 0.18571  10 0.203312 0.217288 0.191108 0.185759 0.2707230.119897 20 0.539455 0.239807 0.140418 0.174014 0.320869 0.094186 301.679507 1.020126 0.584908 0.229946 0.474142 0.154025 40 12.69543 5.9829  7.008946 1.308281 0.301803 0.067526 60 1.271189 2.4029893.186707 5.576734 1.343239 0.115366 100 

[0292] U94-1 U94-2 U94-3 Ratio 0.233894 0.127165 0.804911 10 0.1798551.35532 13.53974 20 0.275078 16.26098 20.65427 30 1.161574 19.9392213.08098 40 1.961067 18.39299 9.319949 60 13.0485 7.591092 1.647718 100

[0293] C32-1 C32-2 C32-3 C32-4 C32-5 C32-6 Ratio 0.137436 0.5441410.138034 0.112832 0.087552 0.131699 10 0.159782 28.93062 14.3276 0.316178 0.125792 0.242881 20 0.166661 53.90695 24.83791 0.67551 0.193545 0.181321 30 0.392402 90.62006 49.11244 2.271509 0.5631680.632798 40 6.034825 73.59378 46.31   2.490156 0.111248 0.273411 6038.17463  60.21433 51.86994 16.43407  2.01284  2.619288 100 

[0294] F32-1 F32-2 F32-3 F32-4 F32-5 F32-6 Ratio 0.746563 2.4466041.288067 0.210478 0.202798 0.112283 10 20.84138 20.94165 11.469631.780569 10.90572  0.100889 20 23.8042 23.7095 13.34488 5.01115 9.510119 0.255589 30 17.47681 17.35353 14.74619 8.361793 6.4363930.599084 40 10.54807 11.78762 13.58168 7.499322 4.865577 0.322946 600.072034 0.090408 0.332458 2.300951 2.434663 1.644695 100 

[0295] F28-1 F28-2 F28-3 F28-4 F28-5 F28-6 Ratio 0.245612 0.2474920.140455 0.203674 0.09426  0.131075 10 0.464885 0.192584 0.2177770.213391 0.171565 0.397716 20 0.290643 0.19239  0.396845 0.4339550.361789 2.02073  30 0.325066 0.189405 1.048323 2.088649 3.88870519.95507  40 0.108766 0.164709 13.95859  0.411927 7.851029 21.77709  600.163978 6.619239 6.832291 8.409421 6.682506 2.958283 100  F28-7 F28-8F28-9 F28-10 F28-11 F28-12 Ratio 0.094505 0.043474 0.05224  0.0503840.104016 0.149513 10 0.173987 0.098512 0.069287 0.057704 0.1310670.028219 20 0.705917 0.254424 0.083005 0.04454  0.133842 0.001619 302.860034 0.928959 0.226468 0.076503 0.095093 0.003896 40 7.7867551.932506 0.42898  0.028744 0.063298 0.001342 60 8.655579 12.729  1.396803 0.281853 0.115513 0.001145 100 

[0296] JJ28-1 JJ28-2 JJ28-3 JJ28-4 JJ28-5 JJ28-6 Ratio 0.122351 0.056864 0.030798 0.041065 0.02373  0.051849 10 0.060598 0.059073 22.465752.229717 0.076134 0.053754 20 0.174243 0.211589 21.92396 4.0895330.121043 0.115906 30 0.133603 36.42899  69.60415 19.16868  0.2922150.337099 40 1.011778 64.69601  71.50927 47.49171  0.755335 0.654333 6060.46546  56.7025   53.44758 39.13032  25.81403 3.936471 100 

[0297] JJ36-1 JJ36-2 JJ36-3 3J36-4 JJ36-5 JJ36-6 Ratio 0.506359 1.6346490.146132 0.053268 0.035023 0.033605 10 2.185596 4.332834 0.6778530.017846 0.010687 0.004264 20 2.339652 5.039758 0.773873 0.0241640.06932  0.009318 30 0.681878 1.871844 1.539743 0.087428 0.0178860.009752 40 0.521703 1.592328 2.000554 0.201203 0.027165 0.004975 600.003277 0.067895 1.031285 0.284902 0.159879 0.008844 100 

[0298] JJ32-1 JJ32-2 JJ32-3 JJ32-4 JJ32-5 J332-6 Ratio  0.392821 1.158486  0.191533 0.127891 0.099083 0.076569 10 17.51289 21.24103 1.803172 0.065286 0.160362 0.108887 20 38.08705 43.77517 24.769270.09612  0.063692 0.044659 30 25.9567  34.88211 26.36994 0.2019070.015214 0.026165 40 11.37519 17.48944 29.59326 0.175101 0.0523210.098545 60 1.3311  1.288845  6.86144 0.70071  0.178921 0.70361  100 

[0299] LL6-1 LL6-2 LL6-3 LL6-4 LL6-5 LL6-6 Ratio 0.405305 0.5834160.520853 0.50183 0.656802 1.060478 10 0.411758 0.747731 0.4600750.287671 0.382454 1.099616 20 0.809416 0.377302 1.253481 1.0999762.59164 1.138122 30 0.475903 0.854576 1.812577 2.018906 2.837056 0.6929840 0.139647 0.29034 0.939013 1.992525 2.250511 3.059824 60 0.001540.002408 0.223682 2.932931 3.939451 6.879564 100

[0300] LL8-1 LL8-2 LLS-3 LL8-4 LL8-5 LL8-6 Ratio 0.533009 1.1808151.581011 2.254195 1.73015 1.76882 10 1.174539 1.228513 1.002632 2.3699431.958308 2.928439 20 1.182611 1.620962 3.771897 3.988759 3.9361240.000474 30 1.366191 1.875091 4.594308 4.253834 4.07168 0.000948 400.120086 0.866135 1.925861 4.423822 4.081074 5.083137 60 0.0033160.029499 0.336777 4.077347 4.416413 4.241522 100

[0301] U28-1 U28-2 U28-3 U28-4 U28-5 U28-6 Ratio 0.049477 0.0444650.045254 0.034669 0.031628 0.025942 10 0.111915 0.050661 0.039880.015399 0.049004 0.020729 20 0.041895 0.050582 0.048212 0.06491710.069067 0.02756 30 0.122271 0.078429 1.647405 0.4885611 1.0362160.058913 40 0.059982 0.051095 18.03734 5.718868 1.991446 0.176516 600.059585 44.91463 51.17075 34.01612 32.39362 3.488256 100

[0302] E28-1 E28-2 E28-3 E28-4 E28-5 E28-6 Ratio 0.109892 0.1500780.630192 0.187585 0.311968 0.168724 10 0.088189 0.509429 0.5893770.106743 0.70723 0.144608 20 1.672278 12.4166 21.41889 3.405963 8.3373411.32881 30 5.658186 12.17055 13.3351 7.272109 17.92266 5.089686 406.016333 5.424512 5.549474 5.185252 15.62457 8.706483 60 1.1460980.804227 0.592348 0.529551 1.752402 5.438448 100

[0303] U36-1 U36-2 U36-3 U36-4 U36-5 U36-6 Ratio 0.158334 0.3883250.255439 0.139618 0.069735 0.054654 10 0.281155 2.119615 0.348030.196109 0.111805 0.067406 20 0.968904 2.501669 3.74982 0.3708140.103744 0.077397 30 0.559332 1.595139 4.650123 0.65127 0.0783430.029797 40 0.565322 0.540675 4.182981 1.59494 0.250723 0.029297 600.238507 0.008686 1.052448 2.269889 1.310025 0.23654 100

[0304] E36-1 E36-2 E36-3 E36-4 E36-5 E36-6 Ratio 0.130066 2.9407340.350723 0.077836 0.051293 0.018123 10 0.496911 1.866482 2.2362570.135236 0.063655 0.020018 20 1.044698 0.617286 4.27308 0.8827250.187495 0.01532 30 0.342085 0.025849 1.935655 1.170393 0.23494 0.00489640 0.112427 0.211108 1.068847 1.362371 0.628612 0.070175 60 0.0025660.003672 0.131476 1.367514 1.194649 0.11883 100

[0305] U32-1 U32-2 U32-3 U32-4 U32-5 U32-6 Ratio 0.202681 0.107654 0.035536 0.037195 0.041027 0.047701 10 0.106511 0.084192  0.0673270.012951 0.028982 0.012003 20 1.497135 2.867785  1.828273 0.0565860.033682 0.010305 30 4.411592 13.8534  0.272404 0.056588 0.0293770.021045 40 17.28347 38.37457  3.026925 0.017452 0.015556 0.060968 6011.81885 13.6584 15.23297 0.673546 0.121004 0.15261 100

[0306] E32-1 E32-2 E32-3 E32-4 E32-5 E32-6 Ratio 0.481414 0.8986990.149685 0.093788 0.086886 0.046001 10 5.170892 6.057429 1.521060.054373 0.099557 0.01378 20 0.965091 2.279423 4.380769 0.1121590.027166 0.038894 30 0.848062 1.086906 3.971834 0.13767 0.0682360.090555 40 0.225141 0.688091 2.653561 0.570809 0.080796 0.01603 600.046762 0.176583 0.897883 1.365759 0.845009 0.111133 100

[0307] C94-1 C94-2 C94-3 C94-4 Ratio 0.289113 0.086166 0.151651 0.20311910 0.133487 0.045908 0.067867 7.650297 20 0.293536 0.086328 0.85331910.31612 30 0.198737 0.170611 1.955433 9.745005 40 0.312808 0.9089913.536115 3.580573 60 0.32801 0.853063 2.414853 1.731594 100

[0308] D94-1 D94-2 D94-3 D94-4 D94-5 D94-6 Ratio 0.223798 0.0912259.083876 0.272732 0.46751 5.545365 10 9.682036 15.16589 24.6553425.45656 27.30727 25.52283 20 14.53736 22.28715 29.68042 38.1211242.28773 33.35092 30 9.804481 14.97104 18.63768 28.87773 35.6740128.4263 40 6.36291 11.60176 16.02556 27.2195 29.006 16.62105 60 2.6819422.585502 3.03267 9.218975 12.48001 9.63693 100

Other Embodiments

[0309] The foregoing has been a description of certain non-limitingpreferred embodiments of the invention. Those of ordinary skill in theart will appreciate that various changes and modifications to thisdescription may be made without departing from the spirit or scope ofthe present invention, as defined in the following claims.

What is claimed is:
 1. A compound of the formula:

wherein X is methyl, OR or NR₂; R is selected from the group consistingof hydrogen, alkyl, alkenyl, alkynyl, cyclic, heterocyclic, aryl, andheteroaryl; each R′ is independently selected from the group consistingof hydrogen, C₁-C₆ lower alkyl, C₁-C₆ lower alkoxy, hydroxy, amino,alkylamino, dialkylamino, cyano, thiol, heteroaryl, aryl, phenyl,heterocyclic, carbocyclic, and halogen; n is an integer between 3 and10,000; x is an integer between 1 and 10; y is an integer between 1 and10; and derivatives and salts thereof.
 2. The compound of claim 1,wherein the compound is amine-terminated.
 3. The compound of claim 1,wherein the compound is acrylate-terminated.
 4. The compound of claim 1,wherein x is an integer between 2 and
 7. 5. The compound of claim 1,wherein x is
 4. 6. The compound of claim 1, wherein x is 5
 7. Thecompound of claim 1, wherein x is
 6. 8. The compound of claim 1, whereiny is an integer between 2 and
 7. 9. The compound of claim 1, wherein yis
 4. 10. The compound of claim 1, wherein y is
 5. 11. The compound ofclaim 1, wherein y is
 6. 12. The compound of claim 1, wherein R ishydrogen.
 13. The compound of claim 1, wherein R is methyl, ethyl,propyl, butyl, pentyl, and hexyl.
 14. The compound of claim 1, whereinthe structure of the compound is

wherein R is hydrogen, x is 4, and y is
 4. 15. The compound of claim 1,wherein the structure of the compound is

wherein R is hydrogen, x is 6, and y is
 4. 16. The compound of claim 1,wherein the structure of the compound is

wherein R is hydrogen, x is 4, and y is
 5. 17. The compound of claim 1,wherein the structure of the compound is

wherein R is hydrogen, x is 5, and y is
 4. 18. The compound of claim 1,wherein the structure of the compound is

wherein R is hydrogen, x is 5, and y is
 5. 19. The compound of claim 1,wherein the structure of the compound is

wherein R is hydrogen, x is 5, and y is
 6. 20. A compound of theformula:

wherein n is an integer between 3 and 10,000; and derivatives and saltsthereof.
 21. A compound of the formula:

wherein n is an integer between 3 and 10,000; and derivatives and saltsthereof.
 22. A compound of the formula:

wherein n is an integer between 3 and 10,000; and derivatives and saltsthereof.
 23. A compound of the formula:

wherein n is an integer between 3 and 10,1000; and derivatives and saltsthereof.
 24. A poly(beta-amino ester) comprising a bis(acrylate ester)selected from the group consisting of formulae A-PP:


25. A poly(beta-amino ester) of claim 24 comprising a bis(acrylateester) of the formula:


26. A poly(beta-amino ester) of claim 24 comprising a bis(acrylateester) of the formula:


27. A poly(beta-amino ester) of claim 24 comprising a bis(acrylateester) of formula:


28. A poly(beta-amino ester) of claim 24 comprising a bis(acrylateester) of formula:


29. A poly(beta-amino ester) of claim 24 comprsing a bis(acrylate ester)of formula:


30. A poly(beta-amino ester) of claim 24 comprising a bis(acrylateester) of formula:


31. A poly(beta-amino ester) comprising an amine selected from the groupconsisting of the formulae 1-94:


32. The poly(beta-amino ester) of claim 31 comprising an amine of theformula:


33. A poly(beta-amino ester) of claim 31 comprising an amine of formula:


34. A poly(beta-amino ester) of claim 31 comprising an amine of formula:


35. A poly(beta-amino ester) of claim 31 comprising an amine of formula:


36. The poly(beta-amino ester) of claim 31 comprising an amine offormula:


37. The poly(beta-amino ester) of claim 31 comprising an amine offormula:


38. The poly(beta-amino ester) of claim 31 comprising an amine offormula:


39. The compound of claim 1, wherein the compound has a molecular weightbetween 1,000 and 100,000 g/mol.
 40. The compound of claim 1, whereinthe compound has a molecular weight between 2,000 and 40,000 g/mol. 41.A pharmaceutical composition comprising a polynucleotide and a compoundof claim
 1. 42. A pharmaceutical composition comprising nanoparticlescontaining a polynucleotide and a compound of claim
 1. 43. Apharmaceutical composition comprising nanoparticles containing apharmaceutical agent and a compound of claim
 1. 44. A pharmaceuticalcomposition comprising microparticles containing an agent encapsulatedin a matrix of a compound of claim
 1. 45. The pharmaceutical compositionof claim 44 wherein the microparticles have a mean diameter of 1-10micrometers.
 46. The pharmaceutical composition of claim 44 wherein themicroparticles have a mean diameter of less than 5 micrometers.
 47. Thepharmaceutical composition of claim 44 wherein the microparticles have amean diameter of less than 1 micrometer.
 48. The pharmaceuticalcomposition of claim 44 wherein the agent is a polynucleotide.
 49. Thepharmaceutical composition of claim 44 wherein the polynucleotide isDNA.
 50. The pharmaceutical composition of claim 44 wherein thepolynucleotide is RNA.
 51. The pharmaceutical composition of claim 44wherein the polynucleotide is an siRNA.
 52. The pharmaceuticalcomposition of claim 44 wherein the agent is a small molecule.
 53. Thepharmaceutical composition of claim 44 wherein the agent is a peptide.54. The pharmaceutical composition of claim 44 wherein the agent is aprotein.
 55. A composition comprising a poly(beta-amino ester), apolynucleotide, and a co-complexing agent selected from the groupconsisting of cationic polymers, cationic proteins, PLGA, spermine,spermidine, polyamines, polyethyleneimine (PEI), and polylysine(PLL).56. The composition of claim 55, wherein the ratio of co-complexingagent to polynucleotide ranges from 0.1 to 1.2 (w/w/w).
 57. Thecomposition of claim 55, wherein the ratio of poly(beta-amino ester) topolynucleotide ranges from 10 to
 120. 58. A method of synthesizing apoly(β-amino ester), the method comprising steps of: providing a primaryamine or bis(secondary amine); providing a bis(acrylate ester); andreacting the amine and the bis(acrylate ester) under suitable conditionsto form the poly(β-amino ester).
 59. The method of claim 58, furthercomprising the step of screening the polymer for a desiredcharacteristic, wherein the poly(β-amino ester) is not precipitated,purified, or isolated before the step of screening.
 60. The method ofclaim 58, wherein the step of reacting comprises reacting the amine andthe bis(acrylate ester) in an organic solvent.
 61. The method of claim58, wherein the step of reacting comprises reacting the amine and thebis(acrylate ester) in the absence of a solvent.
 62. The method of claim58, wherein the organic solvent is selected from the group consisting ofTHF, diethyl ether, glyme, hexanes, methanol, ethanol, isopropanol,methylene chloride, chloroform, carbon tetrachloride, dimethylformamide,acetonitrile, benzene, DMSO, and toluene.
 63. The method of claim 58,wherein the organic solvent is DMSO.
 64. The method of claim 58, whereinthe concentration of the amine is between approximately 0.01 M and 5 M.65. The method of claim 58, wherein the concentration of the amine isbetween approximately 0.1 M and 2 M.
 66. The method of claim 58, whereinthe concentration of the amine is between approximately 1 M and 2 M. 67.The method of claim 58, wherein the concentration of the bis(acrylateester) is between approximately 0.01 M and 5 M.
 68. The method of claim58, wherein the concentration of the bis(acrylate ester) is betweenapproximately 0.1 M and 2 M.
 69. The method of claim 58, wherein theconcentration of the bis(acrylate ester) is between approximately 1 Mand 2 M.
 70. The method of claim 58, wherein the step of reactingcomprises reacting the amine and the bis(acrylate ester) at atemperature between 0 and 75° C.
 71. The method of claim 58, wherein thestep of reacting comprises reacting the amine and the bis(acrylateester) at a temperature between 20 and 50° C.
 72. The method of claim58, wherein the step of reacting comprises reacting the amine and thebis(acrylate ester) at a temperature between 30 and 60° C.
 73. A methodof encapsulating an agent in a matrix of poly(β-amino esters) to formmicroparticles, the method comprising steps of: providing an agent;providing a poly(P-amino ester); and contacting the agent and thepoly(β-amino ester) under suitable conditions to form microparticles.74. The method of claim 73 wherein the agent is a polynucleotide. 75.The method of claim 74 wherein the polynucleotide is DNA.
 76. The methodof claim 74 wherein the polynucleotide is RNA.
 77. The method of claim73 wherein the agent is a small molecule.
 78. The method of claim 73wherein the agent is a protein.
 79. The method of claim 73 wherein thepoly(P-amino ester) is a compound of claim
 1. 80. The method of claim 73wherein the step of contacting comprises spray drying a mixture of theagent and the poly(β-amino ester).
 81. The method of claim 73 whereinthe step of contacting comprises double emulsion solvent evaporationtechniques.
 82. The method of claim 73 wherein the step of contactingcomprises a phase inversion technique.
 83. A method of screening alibrary of polymers, the method comprising steps of: providing aplurality of polymers, wherein the polymers are not polynucleotides orproteins; and screening the polymers for a desired property useful ingene therapy.
 84. A method of screening a library of polymers, themethod comprising steps of: providing a plurality of poly(βaminoesters); and screening the polymers for a desired property.
 85. Themethod of claim 83, wherein the step of providing comprises synthesizingthe polymers in parallel.
 86. The method of claim 85, wherein the stepof synthesizing comprises synthesizing the polymers in DMSO.
 87. Themethod of claim 85, wherein the step of synthesizing does not includeprecipitating, purifiying, or isolating the polymer before the step ofscreening.
 88. The method of claim 83, wherein the plurality ofpoly(beta-amino ester)s comprises at least 500 poly(beta-amino ester)s.89. The method of claim 83, wherein the plurality of poly(beta-aminoester)s comprises at least 1000 poly(beta-amino ester)s.
 90. The methodof claim 83, wherein the plurality of poly(beta-amino ester)s comprisesat least 1500 poly(beta-amino ester)s.
 91. The method of claim 83,wherein the plurality of poly(beta-amino ester)s comprises at least 2000poly(beta-amino ester)s.
 92. The method of claim 83, wherein the desiredproperty is an ability to bind a polynucleotide.
 93. The method of claim83, wherein the desired property is solubility in an aqueous solution.94. The method of claim 83, wherein the desired property is solubilityin an aqueous solution at a pH lower than
 7. 95. The method of claim 83,wherein the desired property is solubility in an aqueous solution at pH5 and not being soluble in an aqueous solution at pH
 7. 96. The methodof claim 83, wherein the desired property is an ability to bind heparin.97. The method of claim 83, wherein the desired property is an abilityto increase transfection efficiency.
 98. The method of claim 83, whereinthe desired property is useful in tissue engineering.
 99. The method ofclaim 83, wherein the desired property is the ability to support cellgrowth.
 100. The method of claim 83, wherein the desired property is theability to support cell attachment.
 101. The method of claim 83, whereinthe desired property is the ability to support tissue growth.
 102. Amethod of screening a collection of polymers, the method comprisingsteps of: providing a plurality of polymers; providing a polynucleotideincluding a reporter gene; providing cells; contacting each of thepolymers with the polynucleotide resulting in a polynucleotide:polymermixture; contacting the cells with polynucleotide:polymer mixture; anddetermining whether cells are expressing the reporter gene.
 103. Themethod of claim 102, wherein the step of providing a plurality ofpolymers comprises synthesizing the polymers in parallel.
 104. Themethod of claim 102, wherein the polymers are poly(beta-amino esters).105. The method of claim 102, wherein the polymers are polyesters. 106.The method of claim 102, wherein the polymers are polyamides.
 107. Themethod of claim 102, wherein the plurality of polymers comprises atleast 500 different polymers.
 108. The method of claim 102, wherein theplurality of polymers comprises at least 1000 different polymers. 109.The method of claim 102, wherein the reporter gene encodes greenfluorescent protein.
 110. The method of claim 102, wherein the cells aremammalian cells.
 111. The method of claim 102, wherein the cells arebacterial cells.
 112. The method of claim 102, wherein the cells areCOS-7 cells.
 113. The method of claim 102, wherein the ratio of polymerto polynucleotide is 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1,50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, 100:1,105:1, 110:1, 120:1, 130:1, 140:1, 150:1, and 200:1.
 114. The method ofclaim 102, wherein the step of determining comprises quantifying theamount of reporter gene expression.